Haploid inducer line for accelerated genome editing

ABSTRACT

Provided herein are materials and in planta methods for using haploid inducer lines containing a targeted endonuclease to generate transgenic or non-transgenic plants with targeted mutations and/or genomic modifications. Also provided herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/741,002, filed on Dec. 29, 2017, which is a National Stageapplication under 35 U.S.C. § 371 of International Application No.PCT/US2016/040398, having an International Filing Date of Jun. 30, 2016,which claims the benefit of U.S. Provisional Application No. 62/186,913,filed on Jun. 30, 2015, and U.S. Provisional Application No. 62/318,913,filed Apr. 6, 2016. The disclosures of the prior applications areconsidered part of (and are incorporated by reference in) the disclosureof this application.

SEQUENCE LISTING

This document contains a Sequence Listing that has been submittedelectronically as an ASCII text file named “Sequence_Listing.txt.” TheASCII text file, created on Feb. 8, 2022, is 48 kilobytes in size. Thematerial in the ASCII text file is hereby incorporated by reference inits entirety.

TECHNICAL FIELD

This document relates to methods for using a haploid inducer linecontaining a targeted endonuclease to generate doubled haploid plantswith targeted mutations in planta. The methods can be used with, forexample, maize, wheat, oat, barley, triticale, and other species thatutilize haploid inducer lines, as well as for Arabidopsis and otherspecies that can generate haploids using a transgenic haploid inducermethod. The methods can be used to generate transgenic or non-transgenicdoubled haploid plants.

BACKGROUND

Traditional plant breeding strategies have been developed over manyyears to introduce desirable traits into plant species, such asincreased yield, resistance to pests, disease, and/or drought, oradaptation to particular environments and growing conditions. Suchstrategies typically require many successive rounds of crossing, andthus it can take many years to successfully alter a specific planttrait. With the advent of transgenic technologies (also referred to as“molecular breeding”), it became possible to engineer plants withgenomic alterations by introducing transgenic constructs or specificnucleotide sequence alterations, thus providing an additional tool forcrop research and improvement. Genetic modification of plants can beachieved by adding one or more specific genes to a plant, or by knockingdown gene expression (e.g., with RNAi), to produce a desirable trait.Modified plants can be produced relatively quickly, since the majorityof the plant genome is not altered with genetic modification. Togenetically modify a plant by adding a gene, for example, a construct isdesigned to express the gene in the plant—typically by including thegene of interest, a promoter to drive transcription of the gene, and atermination sequence to stop transcription of the gene. The constructcarrying the gene(s) of interest is often accompanied by a selectablemarker (e.g., an antibiotic or herbicide resistance gene) for selectionof transformed plants. The construct may be inserted in the plant genomeusing, for example, Agrobacterium, particle bombardment, or a directmethod such as microinjection. In some cases, a plant virus can be usedto insert a genetic construct into a plant.

Transgenic techniques can have drawbacks, however. For example,transgene insertion into the genome (such as that mediated by particlebombardment) is largely random and can lead to multiple insertions,which can cause difficulties in tracking multiple transgenes present ondifferent chromosomes during segregation. Further, expression of thetransgene can be unpredictable due to its chromosomal location, and insome cases, expression of the transgene is silenced. In addition,production of transgenic plants has proven to be a very controversialtopic, with public opinion often being against the creation oftransgenic varieties—particularly where the varieties in question arecrop plants that will be used as food for human consumption.

Genome editing is another method for using transgenes. In this method, atransgene can be introduced to produce a mutation at specific DNAsequence, and then the transgene is removed from the genome. Forexample, an endonuclease transgene can be inserted into the genome at arandom location and expressed to produce a protein or RNA that targetsand mutates a specific sequence of DNA at a second location in thegenome. The transgene insertion site is most likely not linked with themutated locus. Thus, the transgene can be removed from the genome byoutcrossing of the plant or, if the transgene is not homozygous in theplant line, the transgene can be removed simply by selecting progenythat do not contain the transgene. Thus, a plant line can be producedthat has a mutation at a specific DNA sequence and does not contain atransgene.

Traditional methods of introducing mutations into crop varieties (oftenreferred to as “elite lines”) can be time consuming and costly.Traditionally, transgenic modification utilizes lines that are amenableto transformation, but such lines usually are not agronomicallycompetitive. Thus, the first step in genome engineering typically is totransform an endonuclease transgene into a line that is amenable totransformation to generate the desired mutation(s). Once the line ismutated, it is outcrossed to lines that are agronomically competitive(elite lines). The first crossing between a mutated line and an eliteline generates “F₁” plants that contain half of their DNA from themutated line and half of their DNA from the elite line. To recover theelite line's genetic background with the desired mutation(s), an F₁plant is crossed to the elite line (a process called backcrossing) toproduce a BC₁F₁ plant. The BC₁F₁ contains most of its DNA from the eliteline and only some of its DNA from the mutated line. The process ofbackcrossing is repeated two, three, or more times until a sufficientpercentage of the elite line's DNA composition is recovered. Selectionwith molecular markers can be used to ensure that the desired mutationsare carried through the final backcrossing steps. Each round ofbackcrossing and molecular marker selection adds cost and time to theprocess. Further, if a mutation is desired to be in more than one eliteline, the backcrossing process must be repeated to introduce the desiredmutation into the additional elite lines.

SUMMARY

This document is based, at least in part, on the development of aneffective in planta method for gene targeting that, in a singlegeneration, results in mutated, doubled haploid plants that do notcontain a transgene. The method utilizes a plant haploid inducer stockline containing one or more endonucleases to combine (a) haploidinduction through crosses with (b) targeted DNA double strand breaksengineered by the endonuclease, followed by (c) chromosome doublingprocedures. The plant bearing both the haploid inducer capacity and theendonuclease can simultaneously induce both haploidization and mutation,and is thus referred to as a Haploid Inducer Line for Accelerated GenomeEditing (HILAGE). This gene targeting methodology can producenon-transgenic, doubled haploid individuals without the use ofsubsequent backcrossing procedures, and therefore is likely to havesignificant implications in many areas of plant biology. For example,the technology likely will increase the rate of plant functionalgenetics studies. In some cases, the materials and methods providedherein can be used to produce plants that are non-transgenic for theexogenous endonuclease sequences, but that contain a transgene insertedat a targeted location. The methods provided herein also can be used toengineer improved plant traits, such as increased production ofcommercially valuable compounds, improved flavor profiles, increasedgrain and/or biomass yields, enhanced nutritional quality, increasedresistance and/or tolerance to biotic and abiotic stresses, improvedagronomic characteristics, and improved aesthetic traits.

The methods provided herein can be used in plant species in whichhaploid individuals can be produced through crossing. The benefits ofutilizing a HILAGE line carrying an endonuclease transgene to producedoubled haploid individuals with targeted gene mutations can include,for example, (i) the ability to rapidly produce targeted mutations in agenetic background regardless of the background's transformability; (ii)the generation of targeted mutations in planta avoids slow and costlywhole plant transformation, since no further whole plant transformationis required once the transgene is in the HILAGE stock line; (iii) theretention of minimal or no DNA from the HILAGE line in the resultingplants, such that there is no need for timely and expensive backcrossingof the mutation into the elite line, and no yield drag caused by theinitially transformed line's residual DNA containing non-elite genetics;(iv) the non-transgenic status of the resulting haploid and doubledhaploid plants, at least with regard to the exogenous endonucleasesequence; and (v) the ready scalability of the method by adding moreendonucleases to the HILAGE stock line in order to target more than onegene at a time. In addition to scalability in the number of mutationsgenerated per line, the method also is highly scalable in the number oflines that can be mutated each year. These properties thus contribute toa method that is cost effective and time saving, is easily scalable andwidely deployable, and can be readily incorporated into current breedingmethodologies.

In one aspect, referred to herein as “HILAGE-Mutation” or “HILAGE-MUT,”this document features a method for generating a doubled haploid plantcell having a mutation at or near a selected DNA sequence. In someembodiments, the method can include (a) transforming a haploid inducerline with a nucleic acid encoding a rare-cutting endonuclease togenerate a HILAGE stock line having the nucleic acid stably integratedtherein, wherein the nucleic acid encoding the rare-cutting endonucleaseis operably linked to a promoter that is expressed in plant embryosduring at least the first and second cell divisions after fertilization,and wherein the rare-cutting endonuclease is targeted to the selectedDNA sequence; (b) crossing the HILAGE stock line with a targeted line togenerate an F₁ zygote containing the stably integrated nucleic acid; (c)culturing the F₁ zygote such that (i) the rare-cutting endonuclease isexpressed and cleaves chromosomal DNA at or near the selected DNAsequence, wherein repair of the chromosomal DNA after cleavage resultsin the mutation, and (ii) genome elimination takes place such thatchromosomes from the HILAGE stock line are eliminated, resulting in ahaploid cell; and (d) inducing chromosome doubling in the haploid cellto generate a doubled haploid plant cell containing the mutation. Theplant cell can be from maize, wheat, barley, triticale, Arabidopsis,oat, pennycress, tomato, potato, soybean, or camelina. The rare-cuttingendonuclease can be a transcription activator-like effector (TALE)endonuclease, a CRISPR/Cas-based nuclease, a zinc finger nuclease (ZFN),or a meganuclease. The promoter can be a cauliflower mosaic virusdoubled enhanced 35S promoter, a maize ZmUb1 promoter, or a rice APX,OsCc1, EIF5, R1G1B, PGD1, Act1, or SCP1 promoter. The repair can includehomologous recombination. The mutation can include one or morenucleotide substitutions, additions, or deletions, and/or insertion of atransgenic DNA sequence.

In some embodiments, a HILAGE-MUT method for generating a doubledhaploid plant cell having a mutation at or near a selected DNA sequencecan include (a) transforming a plant cell line with a nucleic acidencoding a rare-cutting endonuclease to generate a transgenic plant cellline having the nucleic acid stably integrated therein, wherein thenucleic acid encoding the rare-cutting endonuclease is operably linkedto a promoter that is expressed in plant embryos during at least thefirst and second cell divisions after fertilization, and wherein therare-cutting endonuclease is targeted to the selected DNA sequence; (b)crossing the transgenic plant cell line to a haploid inducer line togenerate a HILAGE stock line that is homozygous for the nucleic acidencoding the rare-cutting endonuclease and is capable of inducinghaploids upon crossing; (c) crossing the HILAGE stock line with atargeted line to generate an F₁ zygote containing the stably integratednucleic acid; (d) culturing the F₁ zygote such that (i) the rare-cuttingendonuclease is expressed and cleaves chromosomal DNA at or near theselected DNA sequence, wherein repair of the chromosomal DNA aftercleavage results in the mutation, and (ii) genome elimination takesplace such that chromosomes from the HILAGE stock line are eliminated,resulting in a haploid cell; and (e) inducing chromosome doubling in thehaploid cell to generate a doubled haploid plant cell containing themutation. The plant cell can be from maize, wheat, barley, triticale,Arabidopsis, oat, pennycress, tomato, potato, soybean, or camelina. Therare-cutting endonuclease can be a TALE endonuclease, a CRISPR/Cas-basednuclease, a ZFN, or a meganuclease. The promoter can be a cauliflowermosaic virus doubled enhanced 35S promoter, a maize ZmUb1 promoter, or arice APX, OsCc1, EIF5, R1G1B, PGD1, Act1, or SCP1 promoter. The repaircan include homologous recombination. The mutation can include one ormore nucleotide substitutions, additions, or deletions, and/or insertionof a transgenic DNA sequence.

In some embodiments, a HILAGE-MUT method for generating a doubledhaploid plant cell having a mutation at or near a selected DNA sequencecan include (a) crossing a HILAGE stock line with a targeted line togenerate an F₁ zygote containing a stably integrated nucleic acid,wherein the haploid inducer line includes a stably integrated nucleicacid encoding a rare-cutting endonuclease, wherein the nucleic acidencoding the rare-cutting endonuclease is operably linked to a promoterthat is expressed in plant embryos during at least the first and secondcell divisions after fertilization, and wherein the rare-cuttingendonuclease is targeted to the selected DNA sequence; (b) culturing theF₁ zygote such that (i) the rare-cutting endonuclease is expressed andcleaves chromosomal DNA at or near the selected DNA sequence, whereinrepair of the chromosomal DNA after cleavage results in the mutation,and (ii) genome elimination takes place such that chromosomes from theHILAGE stock line are eliminated, resulting in a haploid cell; and (c)inducing chromosome doubling in the haploid cell to generate a doubledhaploid plant cell containing the mutation. The plant cell can be frommaize, wheat, barley, triticale, Arabidopsis, oat, pennycress, tomato,potato, soybean, or camelina. The rare-cutting endonuclease can be aTALE endonuclease, a CRISPR/Cas-based nuclease, a ZFN, or ameganuclease. The promoter can be a cauliflower mosaic virus doubledenhanced 35S promoter, a maize ZmUb1 promoter, or a rice APX, OsCc1,EIF5, R1G1B, PGD1, Act1, or SCP1 promoter. The repair can includehomologous recombination. The mutation can include one or morenucleotide substitutions, additions, or deletions, and/or insertion of atransgenic DNA sequence.

In another aspect, referred to herein as “HILAGE-HomologousRecombination” or “HILAGE-HR,” this document features a method forgenerating a doubled haploid plant cell having a transgenic DNA sequenceinserted at or near a selected DNA sequence. In some embodiments, themethod can include (a) transforming a haploid inducer line with (i) afirst transgenic DNA sequence flanked on both sides by DNA sequenceshomologous to sequences upstream and downstream of the selected DNAsequence, and (ii) a second transgenic sequence that encodes arare-cutting endonuclease, to generate a HILAGE stock line having thefirst and second transgenic DNA sequences stably integrated therein,wherein the transgenic DNA sequence encoding the rare-cuttingendonuclease is operably linked to a promoter that is expressed in plantembryos during at least the first and second cell divisions afterfertilization, and wherein the rare-cutting endonuclease is targeted tothe selected DNA sequence; (b) crossing the HILAGE stock line with atargeted line to generate an F₁ zygote containing the stably integratedtransgenes; (c) culturing the F₁ zygote such that (i) the rare-cuttingendonuclease is expressed and cleaves chromosomal DNA of the targetedline at or near the selected DNA sequence, and the first transgenic DNAsequence is inserted at the site of cleavage, and (ii) genomeelimination takes place such that chromosomes from the HILAGE stock lineare eliminated, resulting in a haploid cell; and (d) inducing chromosomedoubling in the haploid cell to generate a doubled haploid plant cellcontaining the first transgenic DNA sequence. The plant cell can be frommaize, wheat, barley, triticale, Arabidopsis, oat, pennycress, tomato,potato, soybean, or camelina. The rare-cutting endonuclease can be aTALE endonuclease, a CRISPR/Cas-based nuclease, a ZFN, or ameganuclease. The promoter can be a cauliflower mosaic virus doubledenhanced 35S promoter, a maize ZmUb1 promoter, or a rice APX, OsCc1,EIF5, R1G1B, PGD1, Act1, or SCP1 promoter.

In some embodiments, a HILAGE-HR method for generating a doubled haploidplant cell having a first transgenic DNA sequence inserted at or near aselected DNA sequence can include (a) transforming a plant cell linewith (i) a first transgenic DNA sequence that is flanked on both sidesby DNA sequences homologous to sequences upstream and downstream of theselected DNA sequence, and (ii) a second transgenic DNA sequence thatencodes a rare-cutting endonuclease, to generate a transgenic plant cellline having the first and second transgenic DNA sequences stablyintegrated therein, wherein the transgenic DNA sequence encoding therare-cutting endonuclease is operably linked to a promoter that isexpressed in plant embryos during at least the first and second celldivisions after fertilization, and wherein the rare-cutting endonucleaseis targeted to the selected DNA sequence; (b) crossing the transgenicplant cell line to a haploid inducer line to generate a HILAGE stockline that is homozygous for the first and second transgenic DNAsequences and can induce haploids upon crossing; (c) crossing the HILAGEstock line with a targeted line to generate an F₁ zygote containing thestably integrated transgenic DNA sequences; (d) culturing the F₁ zygotesuch that (i) the rare-cutting endonuclease is expressed and cleaveschromosomal DNA of the targeted line at or near the selected DNAsequence, and the first transgenic DNA sequence is inserted at the siteof cleavage, and (ii) genome elimination takes place such thatchromosomes from the HILAGE stock line are eliminated; and (e) inducingchromosome doubling in the haploid cell to generate a doubled haploidplant cell containing the first transgenic DNA sequence. The plant cellcan be from maize, wheat, barley, triticale, Arabidopsis, oat,pennycress, tomato, potato, soybean, or camelina. The rare-cuttingendonuclease can be a TALE endonuclease, a CRISPR/Cas-based nuclease, aZFN, or a meganuclease. The promoter can be a cauliflower mosaic virusdoubled enhanced 35S promoter, a maize ZmUb1 promoter, or a rice APX,OsCc1, EIF5, R1G1B, PGD1, Act1, or SCP1 promoter.

In some embodiments, a HILAGE-HR method for generating a doubled haploidplant cell having a transgenic DNA sequence inserted at or near aselected DNA sequence can include (a) crossing a HILAGE stock line witha targeted line to generate an F₁ zygote containing a stably integratedtransgenic DNA sequence, wherein the HILAGE stock line includes (i) astably integrated first transgenic DNA sequence flanked on both sides byDNA sequences homologous to sequences upstream and downstream of theselected DNA sequence, and (ii) a stably integrated second transgenicDNA sequence that encodes a rare-cutting endonuclease, wherein thetransgenic DNA sequence encoding the rare-cutting endonuclease isoperably linked to a promoter that is expressed in plant embryos duringat least the first and second cell divisions after fertilization, andwherein the rare-cutting endonuclease is targeted to the selected DNAsequence; (b) culturing the F₁ zygote such that (i) the rare-cuttingendonuclease is expressed and cleaves chromosomal DNA of the targetedline at or near the selected DNA sequence, and the first transgenic DNAsequence is inserted at the site of cleavage, and (ii) genomeelimination takes place such that chromosomes from the HILAGE stock lineare eliminated; and (c) inducing chromosome doubling in the haploid cellto generate a doubled haploid plant cell containing the first transgenicDNA sequence. The plant cell can be from maize, wheat, barley,triticale, Arabidopsis, oat, pennycress, tomato, potato, soybean, orcamelina. The rare-cutting endonuclease can be a TALE endonuclease, aCRISPR/Cas-based nuclease, a ZFN, or a meganuclease. The promoter can bea cauliflower mosaic virus doubled enhanced 35S promoter, a maize ZmUb1promoter, or a rice APX, OsCc1, EIF5, R1G1B, PGD1, Act1, or SCP1promoter.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram depicting the steps in a method for generating adoubled haploid plant cell containing a targeted mutation.

FIG. 2A is a diagram depicting the steps in a method for generating aHILAGE stock line that contains an endonuclease transgene and a mutationengineered by the endonuclease.

FIG. 2B is a diagram depicting the steps in a method for using theHILAGE stock line of FIG. 2A to generate a doubled haploid elite linethat is homozygous for a mutation engineered by an endonuclease and doesnot include an endonuclease transgene. The haploid inducer stock line inthis diagram is depicted as the female parent in the cross. For haploidinduction in certain species, however, the haploid inducer stock line isused as the male parent in the cross.

FIG. 3 is a diagram depicting the steps in a procedure for inserting atransgene into a targeted line's genome by crossing a HILAGE stock linewith a targeted line. (1) shows the endonuclease target site and thetransgene to be inserted. The HILAGE line DNA strand is flanked by lightgrey tips, while the targeted line's DNA strand is flanked by blacktips. The HILAGE stock line contains both an endonuclease transgene(“Transgene A”) to generate a double strand break in the targeted line'sDNA at a selected location, and a transgene to be inserted (“TransgeneB”) into the targeted line at the location of the double strand break.The transgene is flanked on each side by sequences identical tochromosomal sequences surrounding the endonuclease target site. Theidentical sequences are represented by two light grey lines. In (2), adouble strand break (DSB) in the targeted line's chromosome is createdby the HILAGE stock line's endonuclease (“Transgene A”). In (3), thetargeted line conducts homologous recombination (HR) repair using theHILAGE line's chromosome as the template strand. The chromosome templateprovided by the HILAGE line carries the Transgene B to be inserted.Transgene B is effectively integrated into the targeted line'schromosome using HR. In (4), the HILAGE line's chromosomes are lost,resulting in a haploid plant with the genetics matching the targetedline, with a Transgene B inserted at the specific location. The haploidplant can be chromosome doubled to produce a doubled haploid line.

FIG. 4 is a representative GmCas9 nucleotide sequence (SEQ ID NO:1; 4140nt, including a stop codon, optimized for GmCas9).

FIG. 5 is the full-length amino acid sequence (SEQ ID NO:2) of theGmCas9 nuclease (1379 aa), which is encoded by SEQ ID NO:1. The sequenceincludes an 11 amino acid modified nuclear localization signal (NLS; SEQID NO:4, encoded by SEQ ID NO:3) for Arabidopsis at the C-terminus.

FIG. 6 is the nucleotide sequence of a synthetic guide RNA (gRNA)cassette (SEQ ID NO:5). Lowercase text indicates the incorporatedcloning sites, including protective base pairs. The transcriptioninitiation site from the U6 promoter is shown as the bold and underlinedguanine base (i.e., G) (this is also referred to as the first base ofthe gRNA). Underlined text is the primer sequence sites for amplifyingthe complete cassette.

DETAILED DESCRIPTION

In some embodiments, this document provides effective in planta methodsfor gene targeting that, in a single generation, can result in mutated,doubled haploid plants that do not contain a transgene. In someembodiments, the methods include the use of a plant HILAGE stock lineencoding one or more targeted endonucleases to combine haploid induction(through crosses) with targeted DNA double strand breaks engineered bythe endonuclease. Such methods can be used to introduce one or moremutations (e.g., substitutions, deletions, or insertions of one or morenucleotide bases) into a targeted plant line, and chromosome eliminationand subsequent doubling procedures then can be used to generate doubledhaploid plants. The methods provided herein can be used to producenon-transgenic (at least with respect to the exogenous endonucleasesequence), doubled haploid individuals without the use of subsequentbackcrossing procedures.

Haploid plants contain half of the usual genomic content. Most, but notall, agronomic crop plants are diploid in that they have two completesets of chromosomes, one from each parent. For the sake of thisdisclosure, it can be assumed that the species of interest are diploid,although it also is to be noted that the methods and materials describedherein can be applied to polyploid species that have more than two setsof chromosomes. One method for generating haploid plants involvescrossing a female parent with a haploid inducer male parent, whichresults in a haploid embryo with maternally inherited chromosomes.Alternatively, paternal haploid plants can be generated by crossing amale parent with a haploid-inducer female parent, which results in ahaploid embryo with paternally inherited chromosomes. Haploid embryosand subsequent plants typically are smaller in size than diploid plants,and usually can be easily identified visually. Haploid plants can growto maturity, but are generally sterile. Homozygous diploid plants can beproduced from haploid plants by doubling of chromosomes from the haploidtissue through exposure to an agent such as colchicine, nitrous oxidegas, heat, or trifluralin. See, e.g., Wan et al., Theor Appl Genet,77:889-892, 1989; and U.S. Publication No. 2003/0005479, which areincorporated herein by reference in their entirety. Chromosome doublingcan produce completely homozygous diploid plants, referred to as doubledhaploids. Doubled haploid plants can be fertile, and can perform as anormal diploid plant.

Haploid cells and chromosome doubling can be utilized in combinationwith a targeted endonuclease to generate plants having mutationsengineered at one or more selected positions. One such method isdepicted in FIG. 1 . In this method, haploid pollen is transformed witha transgene encoding an endonuclease targeted to cleave the pollen'schromosomal DNA at a select site. The endonuclease is expressed in thepollen, leading to a chromosomal break at the site targeted by theendonuclease, thus generating a de novo mutation. Through chemical orspontaneous haploid doubling, the cell becomes doubled haploid.

While such methods result in a homozygous mutation at the target site,they also result in a homozygous transgene, which must be removed byoutcrossing or backcrossing. In addition, outcrossing and backcrossing,or additional transformation followed by outcrossing to remove thetransgene, are required to introgress the allele in elite lines.

In contrast, the methods provided herein utilize haploid inducers,endonucleases, and chromosome doubling techniques to efficiently producehomozygous, non-transgenic (at least with regard to the exogenousendonuclease sequence) plants that contain mutations at one or more lociand do not contain DNA from the HILAGE stock line. The HILAGE stock linecarrying one or more transgenes encoding one or more endonucleases iscrossed to one or more lines (referred to herein as “targeted lines”) inwhich one or more targeted gene mutation(s) are desired. In someembodiments, a targeted line is an elite line. The HILAGE stock line'schromosomes are eliminated by the haploid induction process, resultingin a haploid line that only contains the DNA from the targeted line.Before the HILAGE stock line's chromosomes are eliminated, theendonuclease encoded by the transgene(s) in the HILAGE stock line causesmutations at the target location(s) in the targeted line's chromosomes.The plant that results from chromosome elimination is haploid, and hasthe exact genetic composition of the targeted line except for thedesired targeted mutation(s). This haploid plant can be chromosomedoubled to produce a fully inbred line that does not contain theexogenous endonuclease transgene, with the targeted line's genetics andthe desired mutation(s). No backcrossing is needed to introgress thetargeted mutation(s) into the targeted line, and no backcrossing isneeded to remove the endonuclease transgene(s) or DNA derived from theHILAGE stock line.

In some embodiments, the HILAGE-based methods disclosed herein canenable practitioners to achieve high frequencies of gene targeting byusing an endonuclease expressed from a transgene to create a chromosomebreak at a target locus, while simultaneously producing a haploid linethat does not contain the endonuclease transgene or the HILAGE stockline's DNA. To generate a HILAGE stock line, one or more endonucleasetransgenes can be transformed directly into a haploid inducer line.Alternatively, one or more endonuclease transgenes can first betransformed into a line amenable to transformation, and then backcrossedinto the haploid inducer stock line. An exemplary method for generatinga HILAGE stock line is depicted in FIG. 2A. As shown, a haploid inducerline can be transformed with a transgene encoding an endonuclease, whichthen integrates into the genome of the haploid inducer line. Expressionof the endonuclease protein from the transgene leads to targeted DNAcleavage and a de novo chromosomal mutation in the haploid inducergenome. Self-pollination is then used to generate a HILAGE stock linethat is homozygous for the transgene (and also for the de novomutation).

The HILAGE stock line then can be crossed to one or more lines in whichone or more targeted gene mutations are desired (referred to herein as“targeted lines”). An example of this method is depicted in FIG. 2B. Thecross between the two lines results in F₁ embryos, some of which willbecome haploid through the loss of the HILAGE stock line's chromosomes.During the brief time before the HILAGE stock line's chromosomes areeliminated from the embryo, however, the HILAGE stock line'sendonuclease transgene(s) can be expressed and can generate targetedbreaks in the targeted line's chromosomes, thus resulting in targeted denovo mutations in the targeted line's DNA. After subsequent chromosomeelimination, the resulting haploid plant can be chromosome doubled torestore the diploid chromosome number. The resulting line (i) isdiploid, (ii) is non-transgenic (at least for the endonucleasesequence), (iii) contains one or more homozygous targeted mutation(s),(iv) does not contain DNA from another line, and (v) is ready for fieldtesting in one generation.

Haploid inducer lines typically are identified from inter- or specificintra-species crosses, which can result in haploid individuals for acertain percentage of the progeny. For example, in certain species(e.g., maize), haploid induction can be conducted using inter speciescrosses. Some maize lines have a propensity to produce a smallpercentage of haploid progeny when used in crosses. Some species requireintra species crosses or ‘wide’ crosses in order to produce haploids.For example, crosses between (i) wheat and maize, (ii) barley and maize,and (iii) oat and maize result in the elimination of the maizechromosomes and the production of wheat, barley, and oat haploids,respectively. Certain transgenic modifications to the centromere histoneCenH3 gene also have been demonstrated as a means to develop a haploidinducer line. These lines also induce haploidization based on sexualcrosses. In theory, any haploid inducer line that generates haploidsbased on genome elimination following sexual crosses can be developedinto a HILAGE stock by adding an endonuclease transgene that encodes fortargeted modifications. Further, transgenic haploid inductiontechnology, developed as described elsewhere (Ravi and Chan, Nature464(7288):615-618, 2010), involves using a transgenically modifiedArabidopsis plant to produce haploids through crossing.

The endonuclease that generates the targeted chromosome break can be arare-cutting endonuclease such as, for example, a zinc finger nuclease(ZFN), a transcription activator-like effector (TALE) nuclease, ameganuclease, or a CRISPR/Cas system-based endonuclease, as furtherdescribed below.

The transgene encoding the endonuclease can be operably linked to apromoter that is constitutive, cell specific, inducible, or activated byalternative splicing of a suicide exon, provided that the promoter isactivated before chromosome elimination. Suitable promoters include,without limitation, the cauliflower mosaic virus doubled enhanced 35S(CaMV d35S) promoter, the native Arabidopsis 60S ribosomal proteinpromoter, and the native Arabidopsis expansin-like promoter. Typically,a promoter that is useful in the endonuclease constructs provided hereinis one that drives expression in plant embryos during at least one ofthe first few cell divisions (e.g., at least the first or second celldivision, the first and second cell divisions, the first through thirdcell divisions, or first through fourth cell divisions) afterfertilization. In some cases, a promoter that can be used in anendonuclease construct provided herein is one that drives expression ofan encoded endonuclease such that the endonuclease is active on itstarget site(s) present in the target line's chromosome(s) afterfertilization.

As used herein, the term “transgene” or “transgenic DNA sequence” ismeant to include not only sequences encoding polypeptides (e.g.,polypeptides from exogenous species, such as the endonuclease transgenesdescribed herein), but also regulatory sequences such as promotersequences, cisgenes (genetic material from a different line of the samespecies, which may be inserted to, for example, switch out a nativepromoter for a different native promoter, or to add one or moreadditional copies of a native gene), and indeed, any DNA sequence thatis not normally found at the location into which it is to be inserted.

In some embodiments, further transgenes can be added to the endonucleaseconstruct to limit the targeted line's ability to conduct homologousrecombination (HR) or non-homologous end joining (NHEJ), depending onwhether HR or NHEJ is desired. Examples of such transgenes include RNAitransgenes that can be used to decrease expression of particular genesin order to encourage the plant's chromosome double strand break (DSB)repair mechanism in favor of HR or NHEJ (see, Gallego et al., Plant J.35:557-565, 2003; Nishizawa-Yokoi et al., New Phytologist196(4):1048-1059, 20012; and Qi et al., Genome Res. 23:547-554, 2013).For example, decreasing expression of Ku gene homologs (e.g., the riceand Arabidopsis Ku70 and Ku80 genes), Lig4, and/or RAD50 can increasethe rate of HR. See, for example, Jia et al., J. Botany 2012, ID9892722012; Qi et al., supra; Tanaka et al., Biochem. Biophys. Res.Commun. 396:289-293, 2010; Nishizawa-Yokoi et al., supra; and Gherbi etal., EMBO Rep. 2:287-291, 2001). In addition, certain transgenes canincrease HR, including the Escherichia coli recA and ruvC genes, yeastRad54, and homologs of rice ExoI (see, e.g., Reiss et al., Proc. Natl.Acad. Sci. USA 97:3358-3363, 2000; Shalev et al., Proc. Natl. Acad. Sci.USA 96:7398-7402, 1999; and Osakabe and Toki, unpublished results inVoytas, Ann. Rev. Plant Biol. 64:327-350, 2013). Expression of suchtransgenes may be driven by a strong promoter such as, withoutlimitation, 35S (CaMV d35S) or derivatives thereof (e.g., double 35S),ZmUb1 (maize), APX (rice), OsCc1 (rice), EIF5 (rice), R1G1B (rice), PGD1(rice), Act1 (rice), and SCP1 (rice). Alternatively or in addition tothe RNAi transgenes, a HILAGE stock line may carry mutations in theabove mentioned genes in order to promote HR or NHEJ.

In some HILAGE-HR embodiments, a transgenic construct encoding anendonuclease, or a second construct to be combined into the same plantline as the transgenic construct, can contain one or more copies of aDNA sequence having homology to the DNA at and flanking the target site.This sequence of DNA can contain nucleotide changes such as one or morebase pair substitutions, deletions, and/or additions. Alternatively,this sequence may contain a gene, a promoter, a regulatory sequence,and/or a transgene.

In some cases, the HILAGE line can have a mutation at one or more of thesequences targeted by the endonuclease(s). The presence of themutation(s) may increase the likelihood that a mutation is produced inthe resulting haploid individual. If a chromosome break occurs in thetargeted line and the broken chromosome is repaired by HR using theHILAGE stock's chromosome as the template, then the DSB can be“repaired” with the mutation present in the HILAGE stock line.

In some HILAGE-HR embodiments, a HILAGE line can have a secondtransgenic DNA sequence at one or more of the sequences targeted by theendonuclease(s), such that the DSB generated as a result of expressingthe endonuclease from the HILAGE stock can be repaired by integration ofthe second transgenic DNA sequence. See, e.g., FIG. 3 . Further, in someHILAGE-HR embodiments, a DSB can be repaired by HR using the HILAGEgenotype locus as a template, or by using a transgenic sequencecontaining specific nucleotide changes in the HILAGE stock as atemplate, thus resulting in a mutation.

In some embodiments, two or more (e.g., two, three, four, or more thanfour) different or identical endonucleases and/or CRISPR guide RNAs canbe located on separate chromosomes of a HILAGE stock line. Localizingtwo or more endonucleases and/or CRISPR guide RNAs on separatechromosomes may increase the likelihood that one or more of theendonucleases will remain in the plant for a longer period of time,particularly for plants (e.g., oat) in which chromosomes are lost overtime. The longer an endonuclease persists in the plant before beinglost, the greater the chance that the endonuclease will effectivelycause a double stranded break at the target site.

In some cases, multiple loci can be targeted for mutation. It ispossible that in different doubled haploid progeny, only one or a few,but not all, of the multiple target sites will be mutated. Doubledhaploid progeny derived from the same targeted line can be crossedtogether to combine the targeted mutations. Since the doubled haploidindividuals differ only by mutations at the targeted loci, the mutationscan be combined without the need to select on the rest of the genome.For example, if mutations are desired at three target loci (Locus A,Locus B, and Locus C), but only doubled haploid progeny with mutationsat Loci A and B (a/a, b/b, C/C) and Loci A and C (a/a, B/B, c/c) arerecovered, an individual with mutations at Loci A and B (a/a, b/b, C/C)can be crossed to an individual with mutations at Loci A and C (a/a,B/B, c/c) to produce an individual with mutations at all loci (a/a, B/b,C/c). Self-pollinating the F₁ individual (a/a, B/b, C/c) and screeningthe F₂ progeny can result in recovery of the desired individual (a/a,b/b, c/c) with mutations at all three loci.

In some embodiments, a cross can be conducted between an F₁ plant and aHILAGE stock line (rather than by crossing a homozygous parent line tothe haploid inducer stock line). In such embodiments, the doubledhaploid progeny produced will differ for both their genetics and for thepresence or absence of a targeted mutation(s).

Different mutations may be produced, and evaluation of each mutationevent is necessary to determine if the mutation(s) obtained have thedesired result. Mutations that produce a desired phenotype, such asmutations that cause a frame shift and eliminate proper gene function,are referred to herein as “effective mutations” (EM). In some cases,only lines with EM are advanced. In some embodiments, HILAGE-basedmethods are used to add new mutations to a line that already has one ormore EM. This method also can be used to combine two or more EM into asingle line.

In some embodiments, lines with different HILAGE-induced mutations anddifferent genetic backgrounds are crossed together to combine the EM.The resulting progeny can segregate for both the EM and for theirgenetic background.

In some embodiments, through HR, HILAGE-based methods can produceprogeny having the same mutation as the HILAGE inducer line.

In some HILAGE-HR embodiments, an endonuclease construct can be pairedwith a transgene or quantitative trait locus (QTL) to be inserted intothe endonuclease target site in a targeted line. The endonucleaseconstruct and transgene to be inserted can be in the same construct in aHILAGE line or in different constructs in the same HILAGE line. Thetransgene to be inserted into the targeted line's genome can be flankedon each side by DNA sequences homologous to the DNA sequences flankingthe target site of the endonuclease in the targeted line (FIG. 3 ). Suchembodiments can be used in situations in which the transgene ispositioned in the haploid inducer genome at “location B” but it isdesired to move the transgene to “location A” in the DNA of thenon-haploid inducer. In some embodiments, such methods can be used toinsert a transgene at a specific DNA sequence in an elite cultivarwithout having to conduct backcrossing to remove non-elite chromosomematerial from the elite line.

In some embodiments, the endonuclease can be designed to target a genomesequence that is identical to the sequence flanking the transgene to beinserted into a target line after the transgene has been positioned inthe genome. After generation of a DSB at the target site, the plant cellcan undergo HR in order to repair the DSB. The HILAGE line can supplythe DNA template—the transgene flanked by DNA sequences homologous toendogenous DNA sequences flanking the target site. When the targetedline's chromosome break is repaired using the HILAGE line's strandcontaining the transgene, the transgene is effectively inserted into thetargeted line's chromosome at the DSB.

The plants that can be mutated and/or genetically modified and thendouble haploidized according to the methods provided herein can bemonocotyledonous (e.g., maize, barley, wheat, triticale, or oat) ordicotyledonous (e.g., Arabidopsis, potato, tomato, soybean, pennycress,or camelina), as further described below.

Suitable haploid inducer lines can be generated from, for example,maize, barley, wheat, triticale, oat, sorghum, potato, teosinte, andteff. Naturally occurring maize haploid inducer lines can be readilyobtained, as they are used in academia and industry. In someembodiments, the haploid inducer line used in the methods providedherein can be of a species other than maize. In some cases, a haploidinducer line that contains B chromosomes can be used as describedherein, while in other cases, a haploid inducer line that lacks Bchromosomes can be used as described herein. Barley haploids can begenerated by crossing cultivared barley (Hordeum vulgare) to its wildprogenitor species (Hordeum bulbosum). The developing barley embryos canbe grown in tissue culture (a process called embryo rescue) to generatewhole plants. Wheat, triticale, and oat haploids can be generated bypollinating emasculated wheat and triticale spikes and oat panicles withpollen from related species such as, without limitation, maize, sorghum,barley (H. bulbosum), and millet. As with barley, the wheat, triticale,and oat developing embryos must be embryo rescued to generate wholeplants. Haploid plants also can be generated in Arabidopsis, and likelyin other species, using a transgenic haploid inducer line (Ravi andChan, supra). Haploids in potato can be generated by crossing theconventional tetraploid with a diploid Solanum tuberosum gp. Phurejaclone (Peloquin, Hougas and Gabert, Am J Potato 37:289-297, 1960; andHermsen and Verdenius, Euphytica 22(2):244-259, 1973).

Several categories of crosses that can be made to generate haploidinducer lines, including those discussed above, are summarized in TABLE1:

TABLE 1 Maize, sorghum, teiosinte, or teff Wheat Oat Barley TriticaleRye* Wild relative Barley Potato Maize Maize Arabidopsis with CenH3mutation Arabidopsis Sugar beet* Barley* Soybean* Potato* *Possible

As used herein, “plants” and “plant parts” refers to cells, tissues,organs, seeds, and severed parts (e.g., roots, leaves, and flowers) thatretain the distinguishing characteristics of the parent plant. “Seed”refers to any plant structure that is formed by continueddifferentiation of the ovule of the plant, following its normalmaturation point at flower opening, irrespective of whether it is formedin the presence or absence of fertilization and irrespective of whetheror not the seed structure is fertile or infertile.

The term “allele(s)” means any of one or more alternative forms of agene at a particular locus. In a diploid (or amphidiploid) cell of anorganism, alleles of a given gene are located at a specific location orlocus on a chromosome. One allele is present on each chromosome of thepair of homologous chromosomes. “Heterozygous” alleles are two differentalleles residing at a specific locus, positioned individually oncorresponding pairs of homologous chromosomes. “Homozygous” alleles aretwo identical alleles residing at a specific locus, positionedindividually on corresponding pairs of homologous chromosomes in thecell.

“Wild type” as used herein refers to a typical form of a plant or a geneas it most commonly occurs in nature. “Mutant” refers to a plant or agene that includes one or more changes (e.g., nucleotide substitutions,deletions, or additions) in its nucleic acid sequence as compared to thewild type sequence. In some embodiments, a mutation may result in nodetectable amount of functional protein in the plant or plant cell invivo, or may refer to one or more amino acid changes in the proteinproduced. In some embodiments, a mutation can include an insertedtransgene.

“Mutagenesis” as used herein refers to processes in which mutations areintroduced into a selected DNA sequence. Mutations induced byendonucleases generally are obtained by a double strand break, which canresult in insertions or deletions (“indels”) that can be detected bysequencing analysis. Such mutations typically are deletions of severalbase pairs, and have the effect of inactivating the mutated allele.

The term “rare-cutting endonucleases” as used herein refers to naturalor engineered proteins having endonuclease activity directed to nucleicacid sequences having a recognition sequence (target sequence) about12-40 bp in length (e.g., 14-40 or 15-30 bp in length). Typicalrare-cutting endonucleases cause cleavage inside their recognition site,leaving 4 nt staggered cut with 3′OH or 5′OH overhangs. Theserare-cutting endonucleases may be meganucleases, such as wild type orvariant proteins of homing endonucleases, more particularly belonging tothe dodecapeptide family (LAGLIDADG (SEQ ID NO:48); see, WO 2004/067736)or may result from fusion proteins that associate a DNA binding domainand a catalytic domain with cleavage activity. TAL-effectorendonucleases and zinc-finger-nucleases (ZFN) are examples of fusions ofDNA binding domains with the catalytic domain of the endonuclease FokI.Customized TAL effector endonucleases are commercially available underthe trade name TALEN™ (Cellectis, Paris, France). For a review ofrare-cutting endonucleases, see Baker, Nature Methods 9:23-26, 2012.

For example, in some embodiments, mutagenesis can occur via a doublestranded DNA break made by a TAL effector endonuclease targeted to aselected DNA sequence in a plant cell. Such mutagenesis results in “TALeffector endonuclease-induced mutations” (e.g., TAL effectorendonuclease-induced knockouts) and reduced expression of the targetedgene. Methods for selecting endogenous target sequences and generatingTAL effector endonucleases targeted to such sequences can be performedas described elsewhere. See, for example, PCT Publication No. WO2011/072246 (which is incorporated herein by reference in its entirety).TAL effectors are found in plant pathogenic bacteria in the genusXanthomonas. These proteins play important roles in disease, or triggerdefense, by binding host DNA and activating effector-specific host genes(see, e.g., Gu et al., Nature 435:1122-1125, 2005; Yang et al., Proc.Natl. Acad. Sci. USA 103:10503-10508, 2006; Kay et al. Science318:648-651, 2007; Sugio et al., Proc. Natl. Acad. Sci. USA104:10720-10725, 2007; and Romer et al. Science 318:645-648, 2007).Specificity depends on an effector-variable number of imperfect,typically 34 amino acid repeats (Schornack et al., J. Plant Physiol.163:256-272, 2006; and WO 2011/072246). Polymorphisms are presentprimarily at repeat positions 12 and 13, which are referred to herein asthe repeat variable-diresidue (RVD).

The RVDs of TAL effectors correspond to the nucleotides in their targetsites in a direct, linear fashion, one RVD to one nucleotide, with somedegeneracy and no apparent context dependence. This mechanism forprotein-DNA recognition enables target site prediction for new targetspecific TAL effectors, as well as target site selection and engineeringof new TAL effectors with binding specificity for the selected sites.

TAL effector DNA binding domains can be fused to other sequences, suchas endonuclease sequences, resulting in chimeric endonucleases targetedto specific, selected DNA sequences, and leading to subsequent cuttingof the DNA at or near the targeted sequences. Such cuts (i.e.,double-stranded breaks) in DNA can induce mutations into the wild typeDNA sequence via NHEJ or HR), for example. In some cases, TAL effectorendonucleases can be used to facilitate site directed mutagenesis incomplex genomes, knocking out or otherwise altering gene function withgreat precision and high efficiency. The fact that some endonucleases(e.g., FokI) function as dimers can be used to enhance the targetspecificity of the TAL effector endonuclease. For example, in some casesa pair of TAL effector endonuclease monomers targeted to different DNAsequences can be used. When the two TAL effector endonucleaserecognition sites are in close proximity, the inactive monomers can cometogether to create a functional enzyme that cleaves the DNA. Byrequiring DNA binding to activate the nuclease, a highly site-specificrestriction enzyme can be created.

In some embodiments, a rare-cutting endonuclease can be aCRISPR/Cas-based nuclease. In its native context, the CRISPR/Cas systemprovides bacteria and archaea with immunity to invading foreign nucleicacids (Jinek et al. Science 337:816-821, 2012). The CRISPR/Cas system isfunctionally analogous to eukaryotic RNA interference, using RNA basepairing to direct DNA or RNA cleavage. This process relies on (a) smallRNAs that base-pair with sequences carried by invading nucleic acid, and(b) a specialized class of Cas endonucleases that cleave nucleic acidscomplementary to the small RNA. The CRISPR/Cas system can bereprogrammed to create targeted double-strand DNA breaks inhigher-eukaryotic genomes, including animal and plant cells (Mali etal., Science 339:823-826, 2013; and Li et al., Nature Biotechnology31(8): 688-691, 2013). Further, by modifying specific amino acids in theCas protein that are responsible for DNA cleavage, the CRISPR/Cas systemcan function as a DNA nickase (Jinek et al., supra), or as a DNA bindingprotein that has no nuclease or nickase activity but is capable ofinterfering with incoming proteins, including RNA polymerases (Qi etal., Cell 152:1173-1183, 2013).

Directing DNA DSBs, single strand nicks, or binding of the Cas9 proteinto a particular sequence requires CRISPR RNA (crRNA) and tracer RNA(tracrRNA) sequences that aid in directing the Cas/RNA complex to targetDNA sequence (Makarova et al., Nat Rev Microbiol, 9(6):467-477, 2011).The modification of a single targeting RNA can be sufficient to alterthe nucleotide target of a Cas protein. In some cases, crRNA andtracrRNA can be engineered as a single cr/tracrRNA hybrid to direct Casactivity, whether as a nuclease, a nickase, or a DNA binding protein.

In some embodiments, a rare-cutting endonuclease can be a ZNF, which isa fusion that contains engineered zinc-finger domains with the catalyticdomain of a restriction enzyme such as FokI (Porteus and Carroll, NatureBiotechnol 23:967-973, 2005) or a chemical endonuclease (Eisenschmidt etal., Nucl Acids Res 33:7039-7047, 2005; Arimondo et al., Mol Cell Biol26:324-333, 2006; and Simon et al., Biochimie 90:1109-1116, 2008). Inchemical endonucleases, a chemical or peptidic cleaver is conjugatedeither to a polymer of nucleic acids or to another DNA recognizing aspecific target sequence, thereby targeting the cleavage activity to aspecific sequence. Chemical endonucleases also encompass syntheticnucleases like conjugates of orthophenanthroline, a DNA cleavingmolecule, and triplex-forming oligonucleotides (TFOs), known to bindspecific DNA sequences (Kalish and Glazer, Ann NY Acad Sci 1058:151-161,2005). Such chemical endonucleases are comprised in the term“endonuclease” according to the present document. Examples of suchendonuclease include I-Sce I, I-Chu I, I-Cre I, I-Csm I, PI-Sce I,PI-Tli I, PI-Mtu I, I-Ceu I, I-Sce II, I-Sce III, HO, PI-Civ I, PI-CtrI, PI-Aae I, PI-Bsu I, PI-Dha I, PI-Dra I, PI-Mav I, PI-Mch I, PI-Mfu I,PI-Mfl I, PI-Mga I, PI-Mgo I, PI-Min I, PI-Mka I, PI-Mle I, PI-Mma I,PI-Msh I, PI-Msm I, PI-Mth I, PI-Mtu I, PI-Mxe I, PI-Npu I, PI-Pfu I,PI-Rma I, PI-Spb I, PI-Ssp I, PI-Fac I, PI-Mja I, PI-Pho I, PI-Tag I,PI-Thy I, PI-Tko I, PI-Tsp I, and I-MsoI.

The term “expression” as used herein refers to the transcription of aparticular nucleic acid sequence to produce sense or antisense mRNA,and/or the translation of a sense mRNA molecule to produce apolypeptide, with or without subsequent post-translational events.

In some embodiments, expression of the targeted gene can be reduced as aresult of cleavage by the endonuclease. As used herein, “reducing theexpression” of a gene or polypeptide in a plant or a plant cell includesinhibiting, interrupting, knocking-out, or knocking-down the gene orpolypeptide, such that transcription of the gene and/or translation ofthe encoded polypeptide are reduced as compared to a corresponding wildtype plant or plant cell. Expression levels can be assessed usingmethods such as, for example, reverse transcription-polymerase chainreaction (RT-PCR), Northern blotting, dot-blot hybridization, in situhybridization, nuclear run-on and/or nuclear run-off, RNase protection,or immunological and enzymatic methods such as ELISA, radioimmunoassay,and western blotting.

The polynucleotides, vectors, and polypeptides described herein can beintroduced into a number of monocotyledonous and dicotyledonous plantsand plant cell systems, including dicots such as Arabidopsis, potato,tomato, soybean, pennycress, and camelina, as well as monocots such as,corn, barley, wheat, triticale, and oat.

The methods described herein can be utilized with dicotyledonous plantsbelonging, for example, to the orders Magniolales, Illiciales, Laurales,Piperales, Aristochiales, Nymphaeales, Ranunculales, Papeverales,Sarraceniaceae, Trochodendrales, Hamamelidales, Eucomiales,Leitneriales, Myricales, Fagales, Casuarinales, Caryophyllales, Batales,Polygonales, Plumbaginales, Dilleniales, Theales, Malvales, Urticales,Lecythidales, Violales, Salicales, Capparales, Ericales, Diapensales,Ebenales, Primulales, Rosales, Fabales, Podostemales, Haloragales,Myrtales, Cornales, Proteales, Santales, Rafflesiales, Celastrales,Euphorbiales, Rhamnales, Sapindales, Juglandales, Geraniales,Polygalales, Umbellales, Gentianales, Polemoniales, Lamiales,Plantaginales, Scrophulariales, Campanulales, Rubiales, Dipsacales, andAsterales. The methods can be used over a broad range of plant species,including species from the dicot genera Atropa, Alseodaphne, Anacardium,Arachis, Beilschmiedia, Brassica, Carthamus, Cocculus, Croton, Cucumis,Citrus, Citrullus, Capsicum, Catharanthus, Cocos, Coffea, Cucurbita,Daucus, Duguetia, Eschscholzia, Ficus, Fragaria, Glaucium, Glycine,Gossypium, Helianthus, Hevea, Hyoscyamus, Lactuca, Landolphia, Linum,Litsea, Lycopersicon, Lupinus, Manihot, Majorana, Malus, Medicago,Nicotiana, Olea, Parthenium, Papaver, Persea, Phaseolus, Pistacia,Pisum, Pyrus, Prunus, Raphanus, Ricinus, Senecio, Sinomenium, Stephania,Sinapis, Solanum, Theobroma, Trifolium, Trigonella, Vicia, Vinca, Vitis,and Vigna.

The methods described herein also can be utilized with monocotyledonousplants such as those belonging to the orders Alismatales,Hydrocharitales, Najadales, Triuridales, Commelinales, Eriocaulales,Restionales, Poales, Juncales, Cyperales, Typhales, Bromeliales,Zingiberales, Arecales, Cyclanthales, Pandanales, Arales, Lilliales, andOrchidales, or with plants belonging to Gymnospermae, e.g., Pinales,Ginkgoales, Cycadales and Gnetales. The methods can be used over a rangeof species from the monocot genera Allium, Andropogon, Aragrostis,Asparagus, Avena, Cynodon, Elaeis, Festuca, Festulolium, Heterocallis,Hordeum, Lemna, Lolium, Musa, Oryza, Panicum, Pannesetum, Phleum, Poa,Secale, Sorghum, Triticum, and Zea; or the gymnosperm genera Abies,Cunninghamia, Picea, Pinus, and Pseudotsuga.

A plant cell, plant tissue, or whole plant can be identified andisolated by selecting or screening the engineered cells for particulartraits or activities, e.g., those encoded by marker genes or antibioticresistance genes. Such screening and selection methodologies are wellknown to those having ordinary skill in the art. In addition, physicaland biochemical methods can be used to identify transformants. Theseinclude Southern blot analysis or PCR amplification for detection of apolynucleotide; Northern blots, S1 RNase protection, primer-extension,or RT-PCR amplification for detecting RNA transcripts; enzymatic assaysfor detecting enzyme or ribozyme activity of polypeptides andpolynucleotides; and protein gel electrophoresis, Western blots,immunoprecipitation, and enzyme-linked immunoassays to detectpolypeptides. Other techniques such as in situ hybridization, enzymestaining, and immunostaining also can be used to detect the presence orexpression of polypeptides and/or polynucleotides.

Polynucleotides that are stably incorporated into plant cells can beintroduced into other plants using, for example, standard breedingtechniques.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1—HILAGE: Arabidopsis

GmCRISPR Construct Assembly: The GmCRISPR construct consists of a Cas9nuclease and a guide RNA (gRNA). These two genes were assembled inseparate vectors and then combined together into a single vector.

Cas9 Assembly: A Cas9 nuclease was codon optimized to match the primarycodon usage of soybean (Glycine max). The Cas9 nuclease alternativelycould have been designed to match the primary codon usage of Arabidopsisthaliana or another plant or animal species. An Arabidopsis NuclearLocalization Signal (NLS) based on SV40 (Hicks et al., Plant Physiol107(4):1055-1058, 1995) was added to the C terminus of the codonoptimized Cas9 protein. The Cas9-NLS cassette, referred to henceforth asGmCas9 (SEQ ID NO:1, FIG. 4 , encoding SEQ ID NO:2, FIG. 5 ), wassynthesized by Life Technologies (New York). AscI and Pad restrictionsites were present upstream and downstream of the GmCas9 cassette,respectively. The synthesized GmCas9 was cloned into the pmDC32 gatewayvector using digestion with AscI and Pad followed by ligation, whichreplaced a ccdB gene in pmDC32. A NOS terminator was present in thepmDC32 plasmid downstream of the Pad restriction site and upstream of anEcoRI restriction site.

The GmCas9-NOS fragment was transferred from pmDC32 to the destinationvector (pmDC123) by digesting both plasmids with AscI and EcoRI,followed by ligation, to replace the ccdB gene. The pmDC123 destinationvector contains Kanamycin resistance while the gateway vector pmDC32plasmid contains Hygromycin resistance, allowing the use of Kanamycinselection to recover the destination vector. The pmDC123 plasmid alsocontains the BAR gene for resistance to the herbicide bialaphos drivenby the CaMV 35S promoter, making herbicide selection possible afterwhole plant transformation. A cauliflower mosaic virus doubled enhanced35S (CaMV d35S) promoter flanked by HindIII and AscI restriction siteswas added in front of the GmCas9 cassette using digestion with HindIIIand AscI followed by ligation.

Several versions of the pmDC123 GmCas9 cassette were created usingdifferent promoters driving GmCas9. These promoters were PCR amplifiedfrom Arabidopsis thaliana genomic DNA using KOD polymerase (Novozyme,Denmark) and PCR primers designed to contain HindIII and AscIrestriction sites in the forward and reverse primers, respectively. PCRamplicons were double digested with HindIII and AscI and cloned intopmDC123 using the HindIII and AscI restriction sites to replace the d35Spromoter.

TABLE 2 Promoters tested Promoter Arabidopsis # Promoter Gene 1 Doubledenhanced 35S (CaMV d35S) Na 2 Native Arabidopsis 60S Ribosomal AT5G40040protein 3 Native Arabidopsis Expansin-Like AT3G45970 4 NativeArabidopsis Inorganic AT2G38940 Phosphate Transport 1-4 (PHT1; 4)

The PCR primers used to amplify the promoters were:

Expansin-like protein gene promoter: AT3G45970 (SEQ ID NO: 6)F 5′ CCAAGCTTCCCAACTACACGATGGACTCAC 3′  (SEQ ID NO: 7)R 5′ CCGGCGCGCCATGTAAAGAGAAGAGAGGACAAAG 3′ 60S acidic ribosomal protein gene promoter  AT5G40040 (SEQ ID NO: 8)F 5′ CCAAGCTTCCCAACTACACGATGGACTCAC 3′ (SEQ ID NO: 9)R 5′ CCGGCGCGCCATGTAAAGAGAAGAGAGGACAAAG 3′ Native Arabidopsis Inorganic Phosphate Transport 1-4 (PHT1;4)(SEQ ID NO: 10) F 5′ CGAAAATAAATGAAGGCATCAATAAAAGCTTACC 3′ (SEQ ID NO: 11) R 5′ GTCAGCTCGGCGCGCCTCTTCTTCTCCTCTGCAATTTTTCA  TCAC 3′

pBS gRNA Cassette and Vector Assembly: An Arabidopsis thaliana AtU6promoter (AT3G13855) was designed to drive expression of the gRNA (FIG.6 ). A synthetic cassette was generated by Life Technologies (New York)that consisted of an AtU6 promoter and gRNA expression cassette followedby a terminator consisting of eight thymine residues. Both ends of thecassette contained EcoRI restriction sites. This expression cassette wascloned into the pBS vector using EcoRI digestion followed by ligation.The 20 base pair (bp) target sequence fragment was synthesized using apair of 24 bp PCR primers with a 20 bp complimentary sequence. Togenerate the target sequence fragment, the two reverse complimentaryprimers were annealed together in a 1×PCR buffer solution for threehours at anneal temperatures corresponding to the annealing temperaturesof the PCR primers. When combined, the pair of primers generated 4 bp 5′overhangs on each end of the fragment, designed to be compatible withBbsI sites. To insert the target sequence fragment into the gRNA, thevector was digested by BbsI at the two BbsI sites designed inside thetarget sequence 20 bp spacer. Digestion by BbsI removed the 20 bp spacerto allow insertion of the target sequence fragment. Following digestion,the plasmid was treated with Calf Intestinal Phosphatase (CIP; NewEngland Biolabs, MA). The target sequence fragment was then ligated intothe gRNA vector between the BbsI sites.

Sequences of the target oligonucleotides were as follows (with f1/r1being for target 1 and f2/r2 being for target 2):

GL1 gene (AT3G27920.1) (SEQ ID NO: 12) f1 5′ GATTGAGAATCAAGAATACAAGAA 3′(SEQ ID NO: 13) r1 5′ AAACTTCTTGTATTCTTGATTCTC 3′ (SEQ ID NO: 14)f2 5′ GATTGGAAAAGTTGTAGACTGAGA 3′ (SEQ ID NO: 15)r2 5′ AAACTCTCAGTCTACAACTTTTCC 3′

Underlining indicates the 20 bp target sequences.

Combining GmCas9 and gRNA cassettes to form the CRISPR construct: Withthe ccdB site replaced by the GmCas9 cassette, the EcoRI site in thepmDC123 became a unique cut site. The same EcoRI cut sites used toinsert the expression cassette into the pBS vector were used to clonethe gRNA into the pmDC123 vector downstream of the GmCas9 cassette. ThepmDC123 vector, containing the GmCas9 cassette, was digested with EcoRIand CIP treated. The pBS vector also was digested with EcoRI. The gRNAcassette was then ligated into the pmDC123 plasmid between the EcoRIrestriction sites. This construct is referred to going forward as theGmCRISPR construct.

Gus Construct Creation: Several GUS expression constructs were createdto test the efficacy of the selected promoters (TABLE 2). The promoterswere inserted into the pmDC123 plasmid as previously described. The GUSreporter gene was inserted into pmDC123 between the AscI and Pad sites,to replace the GmCas9 fragment without disrupting the NOS terminatorfragment. These constructs are referred to herein as the GUS constructs.

Plant Materials: Columbia (Col) Arabidopsis thaliana seeds homozygousfor the CenH3 GFP-TS-HFD haploid inducer transgene (Ravi and Chan,supra) and segregating for a Single Nucleotide Polymorphism (SNP) inCenH3 (AT1G01370), referred to herein as the CenH3 SNP, were provided byDr. Luca Comai (UC-Davis). Progeny were genotyped for the presence ofthe CenH3 SNP following the methods outlined by Ravi and Chan (supra),who had observed that when outcrossed to wild type Arabidopsis,individuals homozygous for the CenH3 GFP-TS-HFD haploid inducertransgene and homozygous for the mutant CenH3 SNP produced a smallpercentage of haploid individuals not containing the inducer line'schromosomes. The individuals homozygous for the mutant CenH3 SNP weremostly male sterile, and thus, individuals heterozygous for the CenH3SNP change need to be maintained in order to produce more individualswith the homozygous mutant CenH3 SNP. Ler plants with Kanamycinresistance were obtained from Cold Spring Harbor's Gene Trap lines.Plant materials were grown in a growth chamber either in potting mixsoil or in petri dishes on ½MS media with 0.8% agar and 1% sucrose. Thegrowing conditions were 16 hours of light, and temperatures of 22° C.and 20° C. during the day and night, respectively. Plants in soil werefertilized with half strength Hoglands solution every other week.

Plant Transformation: Arabidopsis plants were transformed using thefloral dip method (Clough and Bent, Plant J, 16(6):735-743, 1998). TheGmCRISPR constructs were transformed into plants that were homozygousfor the CenH3 GFP-TS-HFD haploid inducer transgene and genotyped asheterozygous for the SNP change in (AT1G01370). Arabidopsis thaliana gl1gl1 plants were transformed, via floral dip, with the GUS constructs.Arabidopsis thaliana Columbia (Col) plants also were transformed withthe GUS constructs.

Screening T₁ seed for plants containing the GmCRISPR or Gus constructs:After floral dipping, the plants were allowed to mature and set seed.The T₁ seeds were dried at room temperature for 7 days after harvesting.T₁ seeds were planted out in trays filled with potting mix. The soil ofthe flats was misted with water and the flats were covered with a clearlid to maintain higher moisture levels. Ten days after germination, theflat was sprayed with a 0.01% solution of Basta herbicide (Glufosinate)to select individuals containing the pmDC123 constructs. Seventeen (17)days after germination, a second Basta spray was conducted to againselect individuals containing the pmDC123 constructs. Plants thatsurvived both rounds of Basta spray were transplanted to individual potsand assigned a plant identification number. This procedure was used toscreen for plants containing the GmCRISPR construct or the Gusconstructs.

Testing promoters using GUS expression assays: After surviving the Bastaherbicide spray, the T₁ plants containing a GUS construct were grown tothe flowering stage. During flowering, flowers at various stages ofgrowth were excised from the plant and stained following the GUSstaining protocol. The flowers were dissected, and the presence of GUSstaining was assessed under a dissecting microscope. The plants werealso grown out to set seed to maintain the lines.

Screening T₁ CRISPR plants for mutations at the Gl1 locus (AT3G27920):After surviving the Basta herbicide spray, T₁ plants containing aGmCRISPR construct were visually inspected to identify plants not havingtrichomes or having sections of leaves without trichomes. These plantswere genotyped for the presence of mutations at the CRISPR target siteGL1 locus (AT3G27920) (TABLES 3A and 3B). Mutant plant genotypicverification was conducted using CAPS assays as described elsewhere(Curtin et al., Plant Physiol 156(2):466-473, 2011). Briefly, the targetwas PCR amplified using primers flanking the target site. Next, thefragments were digested using the Dde1 restriction enzyme. PCR ampliconsresistant to digestion were submitted for Sanger Sequencing at theUniversity of Minnesota Genotyping Center (UMGC) to confirm the presenceof mutations. It is noted that a decrease in the number of trichomes orthe complete absence of trichomes on the T₁ plants can result fromsomatic mutations, rather than germ line mutations, and thus furtherscreening for mutations at the GL1 locus is required in the nextgeneration.

TABLE 3A  Target Site and Methods for Genotypic Screening EndonucleaseCRISPR Gene Target AT3G27920 Gene Target Gl1 Target sequenceGGAAAAGTTGTAGACTGAGATGG  (SEQ ID NO: 16) Mutant PhenotypeNo trichomes or decreased  trichrome density Genotypic ScreenCAPS assay; enzyme Dde1

TABLE 3B  PCR primers for amplifying CRISPR target site Gene Target Gl1Forward Primer 5′-GCACGTGTCACGAAAACCCATC-3′  (SEQ ID NO: 17)Reverse Primer 5′-ATTGTAGTAACATAAAGTTATGTA-3′ (SEQ ID NO: 18)

Screening of T₁ plants for the CenH3 SNP: Individuals that had theGmCRISPR construct and lacked trichomes or had few trichomes weregenotyped for the CenH3 SNP. The individuals were classified into thethree genotypic classes based on the CenH3 SNP: homozygous wild type,heterozygous mutant, and homozygous mutant.

Selection of T₁ generation GmCRISPR plants to advance: Selections of T₁plants to advance were made based on whether the plant had the GmCRISPR,lacked trichomes or had few trichomes, and was heterozygous for theCenH3 SNP. Thus, T₁ plants genotyped as homozygous wild type for theCenH3 SNP were not advanced. The T₁ plants heterozygous for the CenH3SNP were advanced to maintain seed of the line. No T₁ plants wererecovered that had the GmCRISPR, lacked trichomes, and were homozygousfor the mutant CenH3 SNP.

Selection and advancement of T₂, T₃, and later generations of GmCRISPRplants: T₂ seeds from selected T₁ individuals were either directlyseeded into the soil, or were surface sterilized, grown on ½ MS 0.8%agar media with 1% sucrose in petri dish plates and later transplantedto the soil. Twenty-one days after transplanting to the soil, the plantswere screened for the presence of the GmCRISPR construct using Bastaherbicide, as described above. Next, individuals were visually inspectedfor the absence of trichomes. Segregation ratios of 3:1 (trichomes:notrichomes) were observed, indicating that the GL1 mutation was in thegerm line cells and that the mutation was heritable. At this stage,plants that lack trichomes were expected to result from a plantinheriting a pair of mutant gl1/gl1 alleles, rather than from a plantundergoing somatic mutations causing a gl1/gl1 phenotype. It is notedthat the GmCRISPR also could cause new mutations.

The plants containing the GmCRISPR and lacking trichomes were genotypedfor the CenH3 SNP. Individuals that were homozygous for the wild typeCenH3 SNP (TABLE 4A: Classes 4 and 5) were not advanced. Individualsheterozygous for the CenH3 SNP (TABLE 4A: Classes 6 and 7) were advancedto maintain the plant line. Individuals homozygous for the CenH3 SNP(TABLE 4A: Classes 8 and 9) were selected for preliminary crosses totest the haploid induction and targeted mutagenesis system.

T₃ generation (and later) seeds from selected T₂ individuals were eitherdirectly seeded into the soil or are first surface sterilized, grown on½ MS 0.8% agar media with 1% sucrose in petri dish plates, and latertransplanted to the soil. Twenty-one days after transplanting to thesoil, the plants were screened for the presence of the GmCRISPRconstruct using Basta herbicide sprays. Individual T_(2:3) families weredetermined to be homozygous for the GmCRISPR construct if allindividuals showed herbicide resistance (TABLE 4B: Classes 5, 7, and 9).Only individuals from families homozygous for the GmCRISPR constructwere advanced. Next, individuals were visually inspected for the absenceof trichomes to confirm that they were gl1/gl1. Plants containing theGmCRISPR and lacking trichomes were genotyped for the CenH3 SNP.Individuals identified as homozygous for the wild type CenH3 SNP (TABLE4B: Classes 4 and 5) were not advanced, while individuals heterozygousfor the CenH3 SNP (TABLE 4B: Classes 6 and 7) were advanced to maintainthe plant line. Individuals homozygous for the CenH3 SNP (TABLE 4B:Class 9) were selected for crosses to test the haploid induction andtargeted mutagenesis system.

TABLE 4A List of Expected Genotypic Classes of T₂ Plants T₂ IndividualGenotype Selection Action % GL1 Basta Trichome SNP Advancement Classlikelihood CRISPR locus CenH3 Locus Spray Inspection Genotype Decision 1  25% wt/wt Die 2  37.5% +/wt GL1/— Survive Fail 3 18.75% +/+ GL1/—Survive Fail 4 3.125% +/wt gl1/gl1 CenH3/CenH3 Survive Pass Homo Do notadvance WT 5 1.5625%  +/+ gl1/gl1 CenH3/CenH3 Survive Pass Homo Do notadvance WT 6  6.25% +/wt gl1/gl1 CenH3/cenh3 Survive Pass Het Advance toT₃ 7 3.125% +/+ gl1/gl1 CenH3/cenh3 Survive Pass Het Advance to T₃ 83.125% +/wt gl1/gl1 cenh3/cenh3 Survive Pass Homo Seed not fertile,mutant recover genotype in T₃ from Class 6, could try to use forcrossing 9 1.5625%  +/+ gl1/gl1 cenh3/cenh3 Survive Pass Homo Seed notfertile, mutant recover genotype in T₃from Class 7, could try to use forcrossing

TABLE 4B List of Expected Genotypic Classes of T₃ Plants T_(2:3) FamilyT₃ Individual Genotypes Selection Action Basta Spray GL1 Basta TrichomeSNP Advancement or Class Segregation CRISPR locus CenH3 Locus SprayInspection Genotype Cross Decision 4 3:1 live:die +/wt gl1/gl1CenH3/CenH3 ¾ Pass Homo Do not advance, survive WT originated from Class6 5 All survive +/+ gl1/gl1 CenH3/CenH3 all Pass Homo Do not advance,survive WT originated from Class 7 6 3:1 live:die +/wt gl1/gl1CenH3/cenh3 ¾ Pass Het Do not advance if survive have Class 7 plant 7All survive +/+ gl1/gl1 CenH3/cenh3 all Pass Het Advance to survivemaintain crossing line 8 3:1 live:die +/wt gl1/gl1 cenh3/cenh3 ¾ PassHomo Do not advance. survive mutant Could use for crossing, but notpreferred. 9 All survive +/+ gl1/gl1 cenh3/cenh3 all Pass Homo Use forcrossing survive mutant

Phenotypic screening of seed from Cross Types #1, #2, and #3,Endonuclease target GL1: Crosses are conducted to test the effectivenessof combining a haploid inducer system with a CRISPR targeted mutagenesissystem. Ravi and Chan (supra) found that using the haploid inducer asthe female in a cross, rather than as the male, resulting in a higherpercentage of haploids produced from the cross. Thus, for Cross Types #1and #2, the plants used as the female in the haploid inducer cross areCol, (gl1/gl1), homozygous for the mutant CenH3 SNP, heterozygous orhomozygous for the GmCRISPR transgene, homozygous for the GFP-TS-HFDtransgene, and lacking a Kanamycin (KAN) resistance gene (TABLES 4A and4B, Classes 8 and 9). Only 50% of the gametes produced from individualsheterozygous for the GmCRISPR will actually contain the GmCRISPR, while100% of the gametes produced from individuals homozygous for theGmCRISPR will contain the GmCRISPR construct. Thus, it is preferred tocross with individuals homozygous for the GmCRISPR. For crossesconducted with T₂ plants, the genotype of the GmCRISPR is notdetermined; these crosses are conducted with heterozygous or homozygousGmCRISPR plants.

In Cross Type #1 (TABLE 5), Ler Gl1 Gl1 plants lacking a Kanamycinresistant gene are used as the male. In Cross Type #2 (TABLE 6), Ler Gl1Gl1 plants that contain a Kanamycin resistance gene are used as themale. The presence of the Kanamycin resistance gene in Cross #2 allowsfor removal of any self-pollination derived seed when growing the seedfrom crosses on media containing Kanamycin. To test if the haploidinduction and targeted mutagenesis can work using the haploid induce asthe male of the cross, Cross Type #3 (TABLE 7) is conducted using thehaploid inducer as the male and the Ler line, with or without Kanamycinresistance, as the female. It is noted that since the haploid inducerline is mostly male sterile, crossing it as the male may be difficultand result in a low success rate.

Seeds produced from the crosses are surface sterilized and then grown on½ MS 0.8% agar media with 1% sucrose in petri dish plates. Cross Type #1derived plants do not contain Kanamycin resistance and are thus notplanted on media containing Kanamycin. In contrast, all seed producedfrom effective crosses from Cross Type #2 do contain the Kanamycinresistance gene and are planted on media containing 50 ng/ml Kanamycin.Any seed in Cross Type #2 that is produced from self-pollination doesnot have the Kanamycin resistance gene and are killed by the Kanamycinin the media. Cross Type #3 seeds are not plated on media containingKanamycin. Progeny are phenotypically screened for the presence oftrichomes 14-21 days after germination.

Five phenotypic classes are predicted to be present in the progeny ofCross Types #1, #2, and #3 (TABLES 5, 6, and 7, respectively). Seedlingsfrom these crosses are screened for the presence of GL1 mutations andfor the presence of haploidy. Individuals are visually screened for theabsence of trichomes as a preliminary screen for successfully mutationsat the GL1 locus (AT3G27920). To preliminarily screen for haploidy,individuals are visually assessed for growth and vigor, as diploidindividuals grow much faster and larger than haploid individuals (Raviand Chan, supra). Once identified, individuals without trichomes aretransplanted to soil-containing pots and allowed to develop further.Individuals that show slower growth and are smaller also aretransplanted to soil to develop further. Screening is conducted toidentify individuals that are without trichomes and are smaller and growmore slowly, suggesting that they have been mutated and are haploid.

Individuals homozygous for the mutant CenH3 SNP are mostly male sterile(Ravi and Chan, supra), and thus the occurrence of self-pollination ispredicted to be a rare event. Preliminary screening also showed that thefew seed produced from homozygous mutant CenH3 SNP plants have lowviability. Any individual produced from self-pollination in Cross Type#1 is gl1/gl1 diploid and can be screened out by their more vigorousgrowth as compared to haploid individuals. However, individuals that areproduced from self-pollination in Cross Type #2 are killed by theKanamycin in the media. Any individual produced from self-pollination inCross Type #3 is GL1/GL1 and can be screened out by the presence oftheir trichomes. The number of individuals categorized into one of thefive classes, as well as the number of ungerminated seed, are recordedto determine the percent efficacy of HILAGE.

TABLE 5 Expected genotypic classes from Cross Type #1: Endonucleasetarget Gl1 (AT3G27920) Haploid Successful Identification MutationPhenotype Trait: Fast vs Trait: indicates Class Slow Growing (notrichomes) plant is: A Large plant, No trichomes Self- Fast growingpollination* B Large plant, Trichomes Hybrid, not Fast growinghomozygous mutated C Large plant, No trichomes Hybrid, Fast growingmutated D Small plant, Trichomes Haploid***, Slow growing** nothomozygous mutated E Small plant, No trichomes Haploid***, Slowgrowing** mutated Haploid inducer is used as the female plant. Ler plantis used as the male. Ler^(Φ) male plant does not contain KAN resistance.Cross seed is planted on media without Kanamycin. ^(Φ)Other wild typelines could be used as the male, but different SNP assays would need tobe developed to differentiate between the specific wild type line andCol. *Since the haploid inducer line is mostly male sterile,self-pollination is an unlikely event. **Haploid individuals often aresmaller and grow more slowly than diploid individuals (Ravi and Chan,supra). ***Some slow growing plants may be aneuploid rather than fullyhaploid. Haploid individuals can be distinguished from aneuploidindividuals through genotypic analysis.

TABLE 6 Expected genotypic classes from Cross Type #2: Endonucleasestarget Gl1 (AT3G27920) Haploid Successful Identification Mutation Trait:Fast vs Trait: Phenotype indicates Class Slow Growing (no trichomes)plant is: A na na Any self-pollinated* seeds are killed by Kanamycin.Female in cross does not have a Kanamycin resistance gene. B Largeplant, Trichomes Hybrid, not Fast growing homozygous mutated C Largeplant, No trichomes Hybrid, mutated Fast growing D Small plant,Trichomes Haploid***, not Slow growing** homozygous mutated E Smallplant, No trichomes Haploid***, mutated Slow growing** Haploid induceris used as the female plant. Ler plant is used as the male. Ler maleplant does contain KAN resistance. Cross seed is planted on Kanamycincontaining media. *Since the haploid inducer line is mostly malesterile, thus self-pollination is an unlikely event. The use ofKanamycin in the media prevents this class of plant from germinating.**Haploid individuals often are smaller and grow more slowly thandiploid individuals (Ravi and Chan, supra). ***Some slow growing plantsmay be aneuploid rather than fully haploid. Haploid individuals can bedistinguished from aneuploid individuals through genotypic analysis.

TABLE 7 Expected genotypic classes from Cross Type #3: Endonucleasestarget Gl1 (AT3G27920) Haploid Successful Identification Mutation Trait:Fast vs Trait: Phenotype Class Slow Growing (no trichomes) indicatesplant is: A Large plant, Trichomes Self-pollination Fast growing B Largeplant, Trichomes Hybrid, not Fast growing homozygous mutated C Largeplant, No trichomes Hybrid, is mutated Fast growing D Small plant,Trichomes Haploid**, not Slow growing* homozygous mutated E Small plant,No trichomes Haploid**, is mutated Slow growing* Ler plant is used asthe female plant. Haploid induced plant is used as the male. Ler ^(Φ)female plant does or does not contain a KAN resistance gene. Seed isplanted on normal media. ^(Φ)Other wild type lines could be used as themale, but different SNP assays would need to be developed todifferentiate between the additional wild type line and Col. *Haploidindividuals often are smaller and grow more slowly than diploidindividuals (Ravi and Chan, supra). **Some slow growing plants may beaneuploid rather than fully haploid. Haploid individuals can bedistinguished from aneuploid individuals through genotypic analysis.

Genotypic screening of seed from Cross Types #1, #2, and #3,Endonuclease target: GL1: After phenotypic screening, the individuals ofCross Types #1, #2, and #3 that lack trichomes are genotypicallyscreened to confirm the presence of a mutation at the Gl1 locus(AT3G27920) as previously described. The GL1 CRISPR target site of allindividuals in Class E (TABLES 5, 6, and 7), as well as a subset of theClass C individuals, are sequenced to confirm the presence of amutation.

Genotyping individuals to confirm haploidization: Individuals from ClassE (TABLES 5, 6, and 7) and a set of Ler and Col diploid control plantsare genotyped using a custom SNP chip assay to test the haploid,aneuploid, or diploid state of these individuals. The SNP assay isdesigned to test SNPs identified as polymorphic between Col and Ler atmultiple loci across the five chromosomes of Arabidopsis. Aneuploidindividuals appear genotypically heterozygous for Col and Ler at one ormore SNP positions, while F₁ plants appear heterozygous at all SNPpositions, and haploid plants have the Ler genotype at all positions.Additionally, whole genome sequencing is utilized to confirm the haploidstate of select mutated haploid individuals.

Flow cytometry to confirm the occurrence of haploid individuals: Flowcytometry also is conducted to confirm the presence of haploidindividuals. All individuals that are trichomeless and suspected to behaploid are tested with flow cytometry. Some individuals that havetrichomes and are suspected to be haploid, as well as some known diploidindividuals (as controls) also are tested using flow cytometry.

Growing of haploid individuals and treatment of plants with colchicineto double chromosome numbers: Haploid individuals that are identified ashomozygous Ler and also have a mutation at the target locus arechromosome doubled using colchicine before bolting, following methodsdescribed elsewhere (Ravi and Chan, supra). These individuals are grownup in conditions described elsewhere, and seed is harvested.

Example 2—HILAGE: Maize

Haploid inducer methods: Maize (Zea maize) HILAGE method is beingconducted using the standard maize in vivo haploid induction using across with a haploid inducer line, haploid identification techniques,and subsequent chromosome doubling techniques such as, but not limitedto, those described by Prigge and Melchinger (“Production of Haploidsand Doubled Haploids,” in Maize Plant Cell Culture Protocols, Methods inMolecular Biology, Volume 877, pp. 161-172, 2012) and others. Briefly,the in vivo technique of maize haploid induction first requires that across be made between the line to be induced and the haploid inducerline. The inducer is used as either the male or as the female of thecross. In HILAGE-based methods, the haploid inducer is likely used asthe female, but alternatively, HILAGE-based methods are conducted usingthe haploid inducer as the male. Usually, the haploid inducer has adominant purple pigment gene (e.g., R1-nj) that is used to assist inidentifying seeds that are haploid. The seeds of haploid individualshave a purple aleurone, but lack purple pigment in the endosperm(scutellum), indicating that the germline does not contain the haploidinducer chromosomes. Seeds that have a yellow endosperm and a purplealeurone are planted out and grown up to be seedlings. These seedlingshave their chromosome number doubled using colchicine or other methods.The chromosome doubled haploids are grown in a greenhouse and ortransplanted to the field, and the chromosome doubled plants areself-pollinated to produce doubled haploid seed.

Endonuclease transgene and transgenic construct: Maize HILAGE adds thetargeted mutagenesis component to the in vivo haploid induction systemand thus requires an endonuclease. Examples of useful endonucleasesinclude, without limitation meganucleases, ZFNs, TALE nucleases, andCRISPR/Cas-based nucleases. The endonuclease is designed to target Bm3,but an endonuclease can be designed to target nearly any sequence. Theendonuclease(s) are constructed using methods such as, but not limitedto, those described by Sander et al. (Nature Met 8(1):67-69, 2011),Cermak et al. (Nucl Acids Res 39(17):7879, 2011; with correction at NuclAcids Res 39:e82. doi: 10.1093/nar/gkr218, 2011), and Liang et al. (JGenet Genom 41(2):63-68, 2014). An AdH1 intron1 or an HSp70 intron isincluded in the non-translated leader of the endonuclease gene (U.S.Pat. No. 5,593,874) in order to increase gene expression. The promoterused to drive expression of the endonuclease is expressed during earlyembryo development, and can be endogenous or exogenous. Examples areprovided in TABLE 8.

TABLE 8 Examples of promoters 35S (CaMV d35S) or derivatives (e.g.,double 35S) ZmUb1 (maize) APX (rice) OsCc1 (rice) EIF5 (rice) R1G1B(rice) PGD1 (rice) Act1 (rice) SCP1 (rice)

A method for testing potential promoters for driving endonucleaseexpression includes the following steps:

-   -   1. Develop an endonuclease that targets a gene required to make        anthocyanin/purple pigment (e.g., the R1-nj gene).    -   2. Test different promoters in front of the endonuclease coding        sequence, and generate transgenic plants containing the        endonuclease coding sequence linked to the various promoters.    -   3. Pollinate the transgenic endonuclease-containing plant with a        plant having the purple pigment gene (dominant natural trait; do        not use haploid inducers for this test).    -   4. Determine whether different promoter-endonuclease        combinations result in fewer or more seeds that do not develop        purple endosperm.    -   5. Replicate the same promoter across several transgenic events        to control for positional effects of the transgene in the        genome.    -   6. Select promoters that result in a high proportion of F₁        kernels that are lack a purple endosperm, meaning that the        mutation(s) happened early in the development of the endosperm        in all of the developing endosperm cells.    -   7. Alternatively, the test can be done using a haploid inducer        that does not contain a purple pigment gene but does contain the        endonuclease. Cross the haploid inducer to a line with a purple        pigment gene targeted by the endonuclease. Determine whether any        of the seeds have a purple aleurone (indicating a cross rather        than a self-pollination) and lack a purple endosperm. Plant out        seeds having a purple aleurone and lacking a purple endosperm,        and determine whether any of these individuals are haploid.

The endonuclease construct may include a selectable marker (e.g.,herbicide resistance) to assist with recovery of the transgene duringwhole plant transformation and subsequent backcrossing, although aselectable marker is not required for HILAGE-based methods. In somecases, one or more (e.g., two or more, or three or more) endonucleasesand/or CRISPR guide RNAs are combined into a single construct to targetone or sequences of DNA.

Introgression of the endonuclease transgene into the haploid inducer:The next step in maize HILAGE-based methods is the addition of atransgenic endonuclease gene to the maize haploid inducer line. Theendonuclease transgene is added to the haploid inducer line using, e.g.,direct transformation via an Agrobacterium-based method (such as themethod described by Ishida et al., Nature Biotechnol 14(6):745-750,1996) or particle bombardment (such as the method described byGordon-Kamn et al., Plant Cell Online 2(7):603-618, 1990).Alternatively, a line amenable to transformation is first transformedwith the endonuclease transgene, and the resulting line is then crossedto a haploid inducer line. F₁ diploid progeny are screened from thiscross, and these individuals may be backcrossed to the haploid inducerline. This backcrossing process is repeated several times to recover themajority of the haploid inducer's genetics with the addition of theendonuclease transgene. After a sufficient number (e.g., two, three, orfour) of backcrosses are completed, the resulting backcross plant (e.g.,BC₃F₁) plant is self-pollinated to produce BC₃F₂ individuals. Theseindividuals are screened to find individuals that are genetically verysimilar to the haploid inducer line and are homozygous for theendonuclease transgene(s). In the second method, molecular markers maybe used to select backcross individuals that contain the transgene andhigh percentages of the haploid inducer genome. Selected individuals canbe used for the next round of backcrossing to more quickly recover thegenome of the haploid inducer with the addition of the endonucleasetransgene(s). The resulting line that functions as a haploid inducerline and contains the endonuclease transgene is the haploid inducerstock line.

Testing expression of the endonuclease transgene: Following eitherdirect transformation or transformation of another line followed bybackcrossing, several tests are run on the expression of theendonuclease in the haploid inducer stock line. Alternatively,expression tests are conducted before or concurrently with thebackcrossing to select transgenic events with high expression.Specifically, expression assays for RNA and protein expressed from theendonuclease transgene are conducted to ensure that the transgene iscorrectly expressed. Transformation events with higher expression aredesired for HILAGE-based methods. Efficacy of the transgenetransformation event can additionally be assessed by determining ifmutations are detected in the target site(s) of the line. The presenceof mutations is evaluated as described above for Arabidopsis. Eventswith high gene expression and the presence of mutations in the targetsite(s) can be outcrossed to targeted lines to determine whether haploidprogeny with mutations are generated. Desirable haploidinducer-transgenic event combinations produce a high frequency andnumber of haploid progeny with targeted mutations.

Utilization of Maize HILAGE: The haploid inducer is crossed (either asthe male or female) to a targeted line to generate haploid progeny. Itis noted, however, that if the promoter(s) used in the endonucleaseconstruct result in endonuclease expression before fertilization (aswell as during the first couple of cell divisions), the haploid inducerstock line is used as the female. By using the haloid inducer as thefemale, if the endonuclease is expressed in the egg before pollinationand during the first stages of cell development, the endonuclease canimmediately begin mutating the target sequence upon pollination andcontinue mutating the target sequence before the haploid inducer genomeis lost from the cell. In the first stages of mitosis, before thehaploid inducer genome is eliminated, the targeted endonuclease inducestargeted DNA double strand breaks in the DNA from the maize line. Someof these double stranded breaks are incorrectly repaired and a mutationresults. The haploid progeny genomes are doubled before or after theprogeny are screened for the mutation(s). Once the genomes of thesehaploid individuals are doubled, the individuals are grown out andself-pollinated to produce doubled haploid seed. Different mutations maybe produced, and evaluation of each mutation event is necessary todetermine if the mutation(s) obtained have the desired result. Mutationsthat produce a desired phenotype are referred to herein as “effectivemutations” (EM). Only lines with EM are advanced.

HILAGE-based methods may be conducted on all (or many) of the maizelines that a breeder plans to use as parents for breeding. If a breederdevelops populations using lines that have an EM at all targeted loci,the populations do not segregate for the EM. Thus, the breeding effortsare simplified by not having to select for the presence of the EM.

If the haploid inducer stock line is used as the female, the resultinghaploid and doubled haploid will inherit the cytoplasm from the haploidinducer's stock line. If the haploid inducer stock line's cytoplasm isdesirable, the resulting inbred will inherit the desirable cytoplasm.If, however, the line's own cytoplasm is desired and if the haploidinducer is used as the female in HILAGE-based methods, then theresulting doubled haploid with targeted mutations can be backcrossed asthe male to the original line to recover the original cytoplasm. The F₂progeny of the cross shares the same cytoplasm and background genetics,but differs at the one or multiple targeted mutation loci. Selection canbe conducted among the BC₁F₂ individuals to identify individualshomozygous at the desired target loci.

Exemplary target sites and methods for genotypic screening in maize areprovided in TABLES 9A and 10A, while exemplary primers for amplifyingthe target sites are provided in TABLES 9B and 10B. Expected genotypicclasses from the crosses for the two targets are shown in TABLES 11 and12.

Advantages of Maize HILAGE-based methods can include:

-   -   1. The method produces doubled haploid individuals with the        targeted mutation(s) in less than 1 year (using winter        nurseries), without the expense of backcrossing in a desired        targeted mutation into the targeted line.    -   2. Backcrossing is needed to put the endonuclease transgene into        the haploid inducer line, but no subsequent backcross procedures        are required to induce mutations into tens, hundreds, or even        thousands of elite lines (assuming the inducer stock line is        used as the male or assuming the cytoplasm of the inducer stock        line is acceptable).    -   3. If two or more targeted mutations are desired, multiple        endonuclease transgenes may be placed into the inducer line.    -   4. If multiple mutations are desired, the recovered doubled        haploid individuals may not have all of the desired mutations.        Doubled Haploid progeny with single mutations can be crossed        together, and the F₂ progeny can be screened for individuals        that are homozygous for all desired mutations.    -   5. If the inducer is used as the male, the recovered progeny        will have the cytoplasm of the targeted line. If the cytoplasm        of the inducer is desired (for example to obtain male sterile        cytoplasm), the haploid induce can be used as the female. If the        cytoplasm of the targeted line is desired, crosses can be made        between the non-mutated version of the targeted line (as the        female) and the mutated version of the targeted line (as the        male).    -   6. As stated above, the cells in the first stages of mitosis        (before the haploid genome is removed) may try to repair the DSB        in the targeted line's chromosome by HR using the haploid        inducer stock line's chromosome as the template DNA strand.        However, it is likely that the haploid inducer's gene will        already have been mutated by the endonuclease. Thus, if HR        occurs, the cell will ‘repair’ the break incorrectly by using        the mutated inducer stock line's DNA as the template, and a        mutation will occur in the targeted line's DNA. Potentially, if        HR occurs in this way, specific mutations can be induced in the        targeted line.    -   7. HILAGE-based methods may be useful for transgene insertion        without backcrossing. Transgenes can be introduced into a DSB if        the provided template contains the transgene flanked by        sequences that are homologous to the sequences on either side of        the DSB (see, Shukla et al., Nature 459:437-441, 2009). In        HILAGE-based methods, a transgenic event (e.g., to insert an        herbicide gene) approved by the USDA is backcrossed into the        haploid inducer line. An endonuclease gene is used to target the        relative position of the transgene in a non-transgenic line. (In        a line homozygous for the herbicide gene, the endonuclease would        effectively do nothing.) The transgenic event to be inserted        needs to be flanked on both sides by DNA sequences homologous to        the DNA flanking the target site. When the haploid inducer is        crossed to a targeted line that does not have the herbicide gene        (and thus has the targeted site) the endonuclease will cause a        double strand break at the target site. If the targeted line's        DNA is repaired by HR using the haploid inducer stock line's DNA        (and transgene) as the template, the targeted line DNA “repairs”        the double strand break by putting the transgene sequence in the        double strand break site. Thus, HILAGE-based methods may be used        to place transgenes into targeted lines without having to        backcross. Assuming the sequence surrounding the transgene is        exactly the same as the sequence surrounding the transgenic        event certified by the USDA, the two events are arguably        substantially equivalent.

Maize gene to target—Bm3 ZEAMMB73_595664: Sequences of the targetoligonucleotides were as follows (with f1/r1 for target 1 and f2/r2 fortarget 2):

Maize Bm3 gene (ZEAMMB73_595664) (SEQ ID NO: 19)f1 5′ GATTGGGCTCCACCGCCGGCGACG 3′  (SEQ ID NO: 20)r1 5′ AAACCGTCGCCGGCGGTGGAGCCC 3′  (SEQ ID NO: 21)f2 5′ GATTGAACCAGGACAAGGTCCTCA 3′  (SEQ ID NO: 22)r2 5′ AAACTGAGGACCTTGTCCTGGTTC 3′ 

Underlining indicates the 20 bp target sequences.

TABLE 9A  Target Site and Methods for Genotypic Screening, Target 1Endonuclease CRISPR Gene Target ZEAMMB73_595664 Gene Target Maize Bm3Target sequence GGGCTCCACCGCCGGCGACGTGG  (SEQ ID NO: 23)Mutant Phenotype Brown Midrib Genotypic Screen CAPS assay; enzymes BmgBIand MreI

TABLE 9B  PCR primers for amplifying CRISPR target site, Target 1Gene Target Maize Bm3 Forward Primer 5′-CACGGTGCTTGAATTAGTGCG-3′(SEQ ID NO: 24) Reverse Primer 5′-GGTCCCTCCATCTGGCACCG-3′(SEQ ID NO: 25)

TABLE 10A  Target Site and Methods for Genotypic Screening, Target 2Endonuclease CRISPR Gene Target ZEAMMB73_595664 Gene TargetMaize Bm3 gene Target sequence GAACCAGGACAAGGTCCTCATGG  (SEQ ID NO: 26)Mutant Phenotype Brown Midrib Genotypic Screen CAPS assay; enzyme DrdII,BstNI, and PpuMI

TABLE 10B  PCR primers for amplifying CRISPR target site, Target 2Gene Target Maize Bm3 Forward Primer 5′-GGTGGTGGACGAGGAGGC-3′ (SEQ ID NO: 27) Reverse Primer 5′-GTAGCACCAATGATGAGCGAG-3′(SEQ ID NO: 28)

TABLE 11 Expected genotypic classes from cross: Bm3 endonuclease target(ZEAMMB73_595664) Haploid inducer stock line is the female and has thepurple pigment trait (R1-nj), and is crossed to a line without thepurple pigment trait (see, Brink and Williams, Genet 73(2):273-296,1973). Haploid seeds are identified by color traits; seeds that are nothaploid are readily discarded by visual identification. Only seeds ofclasses D and E are planted out. Haploid Successful IdentificationMutation Phenotype Trait: Seed Trait: (brown indicates Class colormidrib) plant is: A Purple aleurone, Green Midrib Self-pollinationPurple scutellum B Purple aleuron, Green Midrib Hybrid, not Purplescutellum homozygous mutated C Purple aleuron, Brown Midrib Hybrid, isPurple scutellum mutated D Purple aleuron, Green Midrib Haploid*, notYellow scutellum* homozygous mutated E Purple aleuron, Brown MidribHaploid*, is Yellow scutellum* mutated *The seeds of haploid individualshave a purple aleurone but lack purple pigment in the endosperm(scutellum), indicating that the germline does not contain the haploidinducer chromosomes.

TABLE 12 Expected genotypic classes from cross: Bm3 endonuclease target(ZEAMMB73_595664) Haploid inducer stock line is the male and has thepurple pigment trait (R1-nj), and is crossed to a line without thepurple pigment trait (Brink and Williams, supra). Haploid seeds areidentified by color; only seed classes D and E are planted out. HaploidSuccessful Identification Mutation Phenotype Trait: Seed Trait:indicates Class color (brown midrib) plant is: A Yellow aleurone, GreenMidrib Self-pollination Yellow scutellum B Purple aleuron, Green MidribHybrid, not Purple scutellum homozygous mutated C Purple aleuron, BrownMidrib Hybrid, is mutated Purple scutellum D Purple aleuron, GreenMidrib Haploid*, not Yellow scutellum* homozygous mutated E Purplealeuron, Brown Midrib Haploid*, is mutated Yellow scutellum* *The seedsof haploid individuals have a purple aleurone but lack purple pigment inthe endosperm (scutellum), indicating that the germline does not containhaploid inducer chromosomes.

Maize Bm3 and surrounding sequence on chromosome 4 (exons are underlined and inuppercase):  (SEQ ID NO: 29)gtcatggatggagccagtgaactgatgattttttccccaccccgcacgcaacagcatgggtgacaacaaccactcccgctgcggttgggcgagcacatctctacgcacttgacactcacgcaaacctaacgcatactagattaatcatcgccaccaactatcggcgacagaaacgatgggccccgcttctataatcacggtgatgaattagtgcgcgcatagtagtgaaaaataatagtgaaaaataagcagtgcgtgttttggtgtggtggttggtgagccgtccggcccaataaaaacccctcgcaccacctcgtccctcttcgtcgcatcgcacgccatcagcagctagcgcgctcctcgagcccagcagagaaaggccggcctacccactctctctctctctctctccagtctccaccggcagcgctaatcgtaatagccATGGGCTCCACCGCCGGCGACGTGGCCGCGGTGGTGGACGAGGAGGCGTGCATGTACGCGATGCAGCTGGCGTCGTCGTCCATCCTGCCCATGACGCTGAAGAACGCCATCGAGCTGGGCCTGCTGGAGGTGCTGCAGAAGGAGGCCGGCGGCGGCAAGGCGGCGCTGGCGCCCGAGGAGGTGGTGGCGCGGATGCCCGCGGCGCCCGGCGACCCCGCCGCCGCGGCGGCCATGGTGGACCGCATGCTCCGCCTGCTCGCCTCCTACGACGTCGTCCGGTGCCAGATGGAGGACCGGGACGGCCGGTACGAGCGCCGCTACTCCGCCGCGCCCGTCTGCAAGTGGCTCACCCCCAACGAGGACGGCGTGTCCATGGCCGCCCTCGCGCTCATGAACCAGGACAAGGTCCTCATGGAGAGCTGgtgagtagtagccgcatcgcatcaaccaccttctacctatctatatccatcacttgttgctgctggcgtgcgcggcatgcatgatgacgagctcgctcatcattggtgctactagtgatttatttcgtccagtaaaattaattaaggtgcgctgctactctactggctgcggctagcacaaggctggaaatagttgttacttgttatacacgatataatatttctctagaacaaaaaagattttttttttataaaaagcaagcaagaaagaaagtgagtgacttcatgtttttcctaaaaaaaagttaggagtgggatggaaaagtcagcaaggaccacttgtttgttgtccactatccatccagtgggtgagacttttttgcgagacggagcactatattattggccgagtectttttctgtatccgcaaaacggcagccgtcgatcgccggacggatcgacggctcacatgagtgtcgagtccaattccaaccacgagggcggcaaggaaaaccatccgtgctggtctggactttttgccaaactccattcagccattcgccgactgaaggtgaatcttcagacagccagattgtttggtgtctagtgtgtgcgaagatggcgtagaaaagactgagagacagttggctcacacagacaagtgacaactgactatagtatctgcctgcctggctgatgctgatagagatggggactatgtcctgtctgtttcttgtatgcgctgatctgattctgatcactgccactctgccagGTACTATCTCAAGGACGCGGTGCTGGACGGCGGCATCCCGTTCAACAAGGCGTACGGGATGACGGCGTTCGAGTACCACGGCACGGACTCGCGCTTCAACCGCGTGTTCAACGAGGGCATGAAGAACCACTCGGTGATCATCACCAAGAAGCTGCTGGACTTCTACACGGGCTTCGAGGGCGTGTCGACGCTGGTGGACGTGGGCGGCGGCGTGGGCGCCACGCTGCACGCCATCACGTCCCGCCACCCGCACATCTCCGGGGTCAACTTCGACCTGCCGCACGTCATCTCCGAGGCGCCGCCGTTCCCCGGCGTGCGCCACGTGGGCGGGGACATGTTCGCGTCCGTGCCCGCCGGCGACGCCATCCTCATGAAGTGGATCCTCCACGACTGGAGCGACGCGCACTGCGCCACGCTGCTCAAGAACTGCTACGACGCGCTGCCGGAAAATGGCAAGGTCATCGTCGTCGAGTGCGTGCTGCCGGTCAACACGGAGGCCACCCCCAAGGCGCAGGGCGTCTTCCACGTCGACATGATCATGCTCGCGCACAACCCCGGCGGCAAGGAGCGGTACGAGCGCGAGTTCCGCGAGCTCGCAAAGGGCGCCGGCTTCTCCGGGTTCAAGGCCACCTACATCTACGCCAACGCCTGGGCCATCGAGTTCATCAAGTGAaccaccgtcgccgcgatgagatggcatggctgccacatgctttgcttgcttggtcctcgtatcgtacgtcgccgtcgtcgtatatctggttattgcgctgctacctcgctgctctcgcgtatgcatgtacttttgcttaattttctttcttcatatcatgcactctggctggcctagac 

Example 3—HILAGE: Wheat

Haploid inducer methods: A wheat (Triticum aestivum or Triticum durum)HILAGE-based method is conducted using the standard wheat in vivohaploid induction using a cross with a maize pollen to pollinate anemasculated wheat spike, embryo rescue in tissue culture, and subsequentchromosome doubling techniques such as, but not limited to, thosedescribed by Knox et al. (Plant Breeding 119:289-298, 2000) and Inagaki(“Double haploid production in wheat through wide hybridization,” inDouble Haploid Production in Crop Plants: A Manual, Maluszynski, Kasha,Forster and Szarejko (Eds.), pp. 53-58, Kluwer Academic Publishers,Dordrecht, Netherlands, 2003). Briefly, the in vivo technique of wheathaploid induction first requires that an emasculated wheat spike. Thefollowing day, the emasculated wheat spike is pollinated with maizepollen. On days 3 and 4 after emasculation the spike is treated with 2,4-dinitrophenylhydrazone or Dicamba (3,6-dichloro-2-methoxybenzoic acid)(Knox et al., supra). Then about 16-19 days after pollination, thedeveloping wheat embryos are removed from the spike and transferred totissue culture. The developing embryo is grown in tissue culture into aplantlet. The plant is eventually transplanted to the greenhouse,treated with colchicine to double the chromosome number and doubledhaploid seed is harvested.

Alternatively, wheat haploid induction can be induced using sorghum,millet, barley (H. bulbosum), or teosinte pollen. The below procedurewill describe the use of maize as the haploid inducer, but maize couldalternatively be substituted for sorghum, millet, barley (H. bulbosum),or teosinte.

Endonuclease transgene and transgenic construct: The wheat HILAGE-basedmethod adds the targeted mutagenesis component to the in vivo haploidinduction system and thus requires an endonuclease. In wheat HILAGE, oneor more of the maize chromosome(s) are carrying an endonucleasetransgene capable of causing targeted double strand breaks in the wheatgenome. Useful endonucleases include, without limitation, meganucleases,ZFNs, TALE nucleases, and CRISPR/Cas-based endonucleases. Theendonuclease is designed to target Tsn1, but an endonuclease can bedesigned to target nearly any sequence. The endonuclease(s) areconstructed using methods such as, but not limited to, those describedby Sander et al. (supra), Cermak et al. (supra), and Liang et al.(supra). The promoter used to drive expression of the endonuclease canbe endogenous or exogenous. High expression of the endonuclease isessential to increase the chance that a targeted mutation is successfulbefore the removal of the maize chromosomes carrying the endonucleasetransgene. Suitable promoters are expressed during early embryodevelopment, and can be endogenous or exogenous. Examples are providedin TABLE 8.

The endonuclease construct may include a selectable marker, such as anherbicide resistance gene, to assist in recovery of the transgene duringwhole plant transformation and subsequent backcrossing. When included,the herbicide resistance selectable marker is operably linked to apromoter with strong expression in maize and/or wheat.

In some cases, the transgenic construct containing the endonuclease or asecond construct combined into the same maize line contains one or morecopies of a sequence of DNA having homology to the DNA at and flankingthe target site. This sequence of DNA may contain nucleotide changessuch as one or more base pair substitutions and/or deletions and/oradditions. Alternatively, this sequence may contain a gene, a promoter,a regulatory sequence, and/or a transgene.

Testing the endonuclease in transgenic wheat: While HILAGE-based methodsdo not use a transgenic wheat line to generate the final product ofdoubled haploid wheat with targeted mutations, it may be beneficial,though not necessary, to test the efficacy of the targeted endonucleaseconstruct in a transgenic wheat line. Wheat transformation could beconducted following techniques such as, but not limited to, thosedescribe by Weeks et al. (Plant Physiol 102(4):1077-1084, 1993) andCheng et al. (Plant Physiol 115(3):971-980, 1997). Transgenic wheat withputative mutations could be checked for targeted mutations using methodssimilar to those described in the Arabidopsis section. An endonucleasewith efficacy at causing double stranded breaks should be utilized forwheat HILAGE-based methods.

Generating a maize line to use for wheat HILAGE: One major differencebetween wheat HILAGE-based methods and normal doubled haploid creationin wheat is that a transgenic maize line is being used for haploidinduction instead of a conventional maize line. As such, a maize line isbeing transformed with the endonuclease construct. The endonucleasetransgene could be added to the haploid inducer using several methodssuch as, but not limited to: agrobacteria methods (such as thosedescribed by Ishida et al., supra) or by particle bombardment (such asthe method described by Gordon-Kamm et al., supra). Since the line usedfor maize transformation likely is not a prolific haploid inducer, itmay be beneficial, though not necessary, to backcross the endonucleasetransgene(s) into a genetic background which has shown high efficacy inwheat haploid induction. The backcross introgression of the transgeneinto a more suitable maize line could be conducted with the assistanceof molecular markers to select for the presence of the endonucleasetransgene as well as to select for the genetic background of therecurrent parent (the suitable maize line) and against the donor parentline (the originally transformed maize line).

Depending on the promoter chosen to drive the endonuclease, theendonuclease is likely to show different expression in the maize linethan in the progeny of the maize-wheat cross. If the gene is expected toexpress in maize, it may be beneficial to assess RNA and proteinexpression of the endonuclease to confirm that the endonuclease isfunctional.

Genotyping of the mutated wheat plants: The plantlets are genotypedbefore or after transplanting to soil to identify (1) if the desiredtargeted mutation(s) occurred, (2) if the wheat plant no longer containsmaize chromosomes, and (3) if the transgene(s) are no longer present.Additionally, potentially different tillers may need to be genotyped asthe plant could be chimeric for one or more targeted mutations. Checkingfor mutation(s) at the target site(s) can be conducted as previouslydescribed in the Arabidopsis section. The presence of maize chromosomes,could be assessed by one or more of several methods. Primers can bedesigned to amplify specific sequences on each of the 10 maizechromosomes in the maize line used for haploid induction, and theseprimers can be used to determine if the maize chromosomes are stillpresent. Alternatively, a custom SNP chip can be designed that can beused to genotyped the wheat line and also maize DNA. In wheat plantsthat have lost the maize chromosomes, the wheat SNPs are able to begenotyped, but the maize SNPs are not able to be genotyped.Alternatively or additionally, a low coverage whole genome sequencingmethod or RNA sequencing method could be utilized to determine if themaize chromosomes are present and/or maize genes are being expressed. Ifthe maize chromosomes have been removed from the wheat plant, it islikely that the transgene had also been removed. However, to increaseindustry and consumer acceptance of HILAGE, it may be beneficial to testfor the absence of the transgene(s) in the wheat line. In one method,primers that amplify portions or all of the transgenic construct can bedesigned and used to test if any portion of the construct is in theproduced wheat line. Alternatively, the sequences of the transgene canbe search for in whole genome sequence or RNA sequence data, if saiddata are available.

Utilization of Wheat HILAGE: The maize line containing one or moreendonuclease and or CRISPR guide RNAs is being crossed (as the pollendonor) to a wheat line to generate haploid progeny. Before the maizechromosomes are eliminated, the targeted endonuclease induces targetedDNA double strand breaks in the DNA from the wheat line. Some of thesedouble stranded breaks will be incorrectly repaired and a mutation willresult. The haploid progeny genomes can be doubled before or after theprogeny are screened for the mutation(s). Once the genomes of thesehaploid individuals are doubled the individuals can be grown out andself-pollinated to produce doubled haploid seed. Different mutations maybe produced, and evaluation of each mutation event is necessary todetermine if it has the desired result. Only lines with EM, whichproduce a desired phenotype (e.g., mutations that cause a frame shiftand eliminate proper gene function), are advanced.

In some cases, HILAGE-based methods are conducted on all (or many) ofthe wheat lines that are to be used as parents for breeding. Ifpopulations are developed using lines that have an EM at all targetedloci, the populations will not segregate for the EM. Thus, breedingefforts are simplified by not having to conduct selections for thepresence of the EM.

Advantages of HILAGE in Wheat: HILAGE could play a pivotal role ingenerating targeted mutations in wheat. Globally, there is stillresistance to utilizing transgenes in wheat. HILAGE could provide amethod to induce targeted mutations in wheat without the released wheatline ever technically coming in contact with a transgene placed into awheat chromosome.

HILAGE-based methods may be more effective in wheat than in maize sinceit is likely that the maize chromosomes persist longer in themaize-wheat embryo than the haploid inducer maize chromosomes persist inthe maize haploid inducer-regular maize line embryo. The additional timethat the maize chromosomes are residing in the wheat embryo, the moreopportunity for targeted mutations to occur.

Exemplary target sites and methods for genotypic screening in wheat areprovided in TABLE 13A, while exemplary primers for amplifying the targetsite are provided in TABLE 13B. Expected genotypic classes from thecross are shown in TABLE 14.

Wheat gene to target—Tsn1: Sequences of the target oligonucleotides wereas follows (with f1/r1 being for target 1 and f2/r2 being for target 2).

Tsn1 gene (SEQ ID NO: 30) f1 5′ GATTGCCGCTAGGGCATCTTAGAT 3′ (SEQ ID NO: 31) r1 5′ AAACATCTAAGATGCCCTAGCGGC 3′ 

Underlining indicates the 20 bp target sequences.

TABLE 13A  Target Site and Methods for Genotypic Screening, Target 1Endonuclease CRISPR Gene Target Wheat Tsn1 Gene Target ADH59425 Target GCCGCTAGGGCATCTTAGATAGG  sequence (SEQ ID NO: 32) Mutant Resistance to Stagonospora Phenotype nodorum, which causes Stagonosporanodorum blotch (SNB); and resistance to Pyrenophora tritici-repentis, which causes tan spot.* Genotypic  CAPS assay; enzymes SfaNI, DdeI, Screen BglI, TauI, and AciI *Faris et al, Proc. Natl. Acad. Sci. U.S.A.107(30): 544-13549, 2010.

TABLE 13B  PCR primers for amplifying CRISPR target site, Target 1Gene Target Wheat Tsn1 Forward Primer 5′-TGTGCATTCTTTCCAAAAGGTCA-3′ (SEQ ID NO: 33) Reverse Primer 5′-GCTCCAAAGGGCTTTAGTAGGA-3′ (SEQ ID NO: 34)

TABLE 14 Expected genotypic classes from cross: Tsn1 endonuclease targetDue to the method of wheat haploidization formed from crossingemasculated wheat spikes with maize pollen, several classes of plantoutcomes are not possible. If the wheat is emasculated correctly, noself- pollinations should occur. If a mistake is made in theemasculation process and a wheat seed is allow to self-pollinate, theseed will grow more vigorously than a wheat x maize cross, and the seedcan be easily screened out. Due to the inability of wheat and maizechromosomes to pair and the inability of maize chromosomes to beinherited, classes B and C are not possible. Thus only classes D and Eshould be produced. Haploid Successful Identification Mutation PhenotypeTrait: embryo Trait: disease indicates Class growth resistance plant is:A Screened out na Self-pollination visual as a healthy seed B NA naHybrid, not homozygous mutated C NA na Hybrid, is mutated D Slow growingSusceptible to Haploid, not embryo SNB homozygous mutated E Slow growingResistant to SNB Haploid, mutated embryo

Wheat Tsn1 and surrounding sequence (GENBANK ® accession number GU259618) (SEQ ID NO: 35)ATGACTACACCAATGAGTATACCGTTCGCAACTTTGGAAAAGATTACAAATGGGTTCTCAAACGATTTAATAATTGGAAGGGGTGGGTATGGAAACGTTTACAAGGTATGGCTTAATACTTGATATTTCCTTTTTTCAGCAAATGTTCAGGCTATAAACAAATAATTTAAGTGCAATAATTATGTCAAGCAGGCAGTTTACAAAGGGGAAGTGATTGCTGTGAAGTTGCTTCATGATGATCTGGTGCAATTACTTGATGACAGACAATTTAAAAATGAACTTTTTAACCTTTTGAGGGTTGAGCATCCGAATATTGTTTGCTTACGTGGTTATTGTTATGAAACACGGTATAAAATTGTTAAGCACAATGGTGAGACAGTCTTTGGTAAACATATACACAGAGTTCTCTGCTTTGAATACTTGGAGGGTGGAAGCCTAGACAATCATCTTCATGGTACGATGGAACTTCAAAATACAGTTATTTTGTTTTACGTTTAAAGGAAACTGATTTCTCATTTACATACATACTCTTTGTTAACTTGCGTAGCACCATCTTTGCCACCTAACTGGACCACACGTTACAATACCATAAAGGGGATTTGTGAAGGCTTAAATTTCCTTCACGGATGTCAACCACCAATTTTGCATCTTGATCTGAAGCCTGCCAATATATTAGTAGACAGTTCCATGGTGCCTAAACTGGCGGATTTTGGATTGTCAAAGCTCTTCCATGGATCACATACTCATGTGACAAAACAAATCATAGGAACCCAGTAAGCGGAAGCGACCCGTGGATTGTCTCGTTCTGAATTTTCTTTCTTTTGTGATCAAATAAATAGTATGTACAGTTCTGTACTAACTGTGTCTTTGTATCACGCAGGAAGTACATGCCACCGGAATTCATCAAAGATGGCAAGATCTCGGTTAAAAATGATGTCTTTAGTTTGGGTATTGTGATCATAGAAATAATGGCAGGACCTATGGGTTATTCAGAATTTTCAGAAATGGGCAGCGGTGCACAATTTGTGAAGGAGGTAATAAAAAAAACTCAAGTTTGACACCCGAGTTCGTATAAATAACAAACTACCACACCAAGAATTTGATGTCTAATGTGTGAGCCATTATAATCGTTGAACTGAGTTTATGACAGGACCGGCAGTAATAAAAAATATAGCAACACTCCCCCACACAATATATTGAGCATAGAAGATACAACTTATCTAGCTATAACAAAATAATAATCCAGAAAAGTAGCCATTTTTTTTTCCGGACAGGATTGAGGTCCACCAGTCCAATAACTATGAAGCAGCTCGCTGATAGAAAATTCCAAGGTACAATTATTTTTGTAAGTTTCTCCTTATCACGTGTGAAACACCAATGTAATAAAGCTGATAAACCAAACGTACCCACTATGAGAACTGCATACACTGAGACTCGAAGAAAAGAACAAATGCATATCTAGAACCTTGCTCCATGGGATATCTAGAACCTTGCTCCATGGGATCTAGCACCATCTCCATTTTGGAGCAAGCACGAGGTGCGTATCGTAATCTTTTTCTGCTAGATGCAGACTTAGACACCCAGTATTCTCTAGGTAAATTATTTATCTGGAAAGTCGTAGGTAACACTTGTGAACAAGGATATAGCGTACATATATATGGGAGCATTTGTGTTATGTGACACTTTTGACTTAATTGCAAATATTATGTTATGTGAAGACTCAAGAGTGTTTTTGAACAAGTATCGTACATATTGTACCGAAAAAGGCTTTCGCCCCGCTTTATATTATAAAGCACATGCCCAAGCCAACAAACCACACAGGTTCACAAACACACGCAGACCCACACACACCAAGTTCACACACAGACAAGATCCACAAGGGTTAATGCTGAGGGCACAGCTTAACAAGCCCTAGAACAAAAAGGAAAGACACCATCTAGTCGGGCTCCGGGGGGGGGGGGGGGGGGGGCGGCGGAAGTGGAGGCGCCAGGCGGAAGGCGAGCGATCGAAGGTCGGCGAGGAGGGTGTTGATGATGTCCCGATCCTGAGGGCGGCTAAGCGGCCGCCAAAGCTGCAAGTACCCACACATTTTAAAAATGGCGTCAGTAGCGCGTCGTAGAGGGACTTTTTGGATGACAAGCTTATTGCGGACGGTCCACAGCGTCCAGCCGAGAACCCCAACGCATAACCAACGGATATGTCGGTGGCGTGGGGGGGGAGGCGTGGATTTCCGCGAGGAGGTCGGGGAAGTTGGAGTTGCACCACTATCCGCCAACCGTCTCACGGAAACTGGACCAAAGAAACTGGCCGCAGGGCACGTGAAGAAGATGTGGTTAGCATCCTCCGCAGTGCCGCACAAGGGGCAAAGCCCATCCCCGGGTCCGTTGCGCTTGAGGACTTCGACACCGGAGGGGAGGCGGCCACGAATCCACTGCCAAAGGAAGATCCTAATCTTCAGAGGTAAGCGAATGTCCCAGATCAGAGCAAAGGGCTCGGGCGCGGGCGAAGGCGCAATAGCCGCGTACATGACCTAGTAGAGAAACGACCGGAGGACTCTAGGCGCCACGAGATGGCGTCCGGGGCGTCGGTGACGCTCATCGGAAGAAGGGCGATGTCCTGGAGGAGGGAATCCCAGGCGGCCACTTCGGGGGGACCGAAAGGACGACGAAACGCGAGGCGCCCTAAGTCAATAAGGGCCGTCTCGACAGAGACCCGAGGGTCAACCGCAATGGTGAAGAGATCGGGAAAGCGGGCGGCCAGAGGGGTGTCACCGAGCCACCGATCAAACCAGAACAGGGTCGCGGACCCAGTACCAATCGAAATGGACGTGCCGATACGAAGCACAGGAAGCAGCCGCACGACGGCCTGCCAAAACTGTGATCCGCCCGAACGCTGACAGAAAGCCAGAGGCTGGCCACGGAGGTATTTGTTGCGGATAATGGTGAGCCACAACCCTCCGTCACCATTGGCAATACGCCACAACCACCGGGTCAGGAGGGCGATGTTCATTCGGCGGGAGGACAGAATCTCAAGACCCCCCTGGTCTTTAGGTTTACAAATGTCCGGCCAAGTCACCATGTGGTACTTCTGTTTGTCATCGTCGCCAGCCCAATAGAACCTGGATTGGTACTTGGCAATTTCCGTGTGCAGCGTTTCATGGAGGCTATAAAAGCTCATGAGGAACCAAAGGAGACTGGCGAGTGAGGAGTTGATGAGGATCACCCGCGCCGCCTTTGATAGCCAACGCCCTTTCCAAGGTTCGACGCGGTGTTGCATACGGGTCACCGTAGGGTGGAGGTCCGCCACGGTGAGGCGCGAGTCACTAACGGGGATCCCCAGGTAGGTCGTGGGGAAGGACCCTAGCCGACAGTTCAGGCGATCAGCAATATCCTGAGCCTCCTCCGGAGGGTATCCAAGGACCATCACCTCGCTCTTATCAAAGTTAATCGTAAGGCCCGACATCTGCTGGAAGCACAGGAGGAGGAACTTCAGGTTAGCAACATCCTGATTTGAACCTTCCACCATTATTATGGTGTCGTTCGCGTATTGCAGGAGGGAGACCCCTCCCCCTCCAACTAGGTGAGGGACAATGCCGTGGATATGGCCAGCACCCTTAGCCTTATCCAGGATGGCGGCCAGAGCATCGACCACCATGTTGAACAGGAACGGCGAGAATGGGTCTCCTGACAGACCCCACAGAGGGTGGGGAAGTATGGCCCAATCTCGCCGTTAATGTTCACCGCCGTCTTTCCACATGAAACTGATTGCATCACGCGGGTCACCCAGCGGTCATCAAAGCCCTTACGCAGCAGTACTTCCCGAAGGAAGGGCCAGTGAACAGTATCATAGGCTTTATGGAAGTCAAGCTTCAGGAACACAGCACGAAGATGCTTCACCCGGACCTCGTGAAGGACTTCATGGAATACCAACACGCCATCAAGAATAAACCGGCCTTGGATGAAGGCCGATTGGTTCGGGTGAGTGATCGAATCAGCCAGCAGGGTCACCCTATTGGCGTACCCTTTGGCCAGGATCCGAAAAATCACGTTAATCACCGTGATGGGGCGGAACTGGCGAATATCAGAGGCACCCGGAACCTTTGGGATGAGGGTAATGATCCCATAGTTGAGGCGTCCCAGGTCCATCGAACCCGAATAGAACTCCTCGAACAAAGCCATGACCTCCGGTTTGACCGCCTGCCAGAATGTTTTAAAGAAAGCAACAGGCAGGCCATCCGGGCCTGGGGCCGAGGCGGGGTTCATGCCTTTAATGGCCGCGAGCACCTCGTCCTCGGCGAAGGGAGCAACCAGGGCCGCATTGGCCTCGCCGGGAACCAACTGCGCCCCCGTCCAAGTATCGGGGGCATCGTACATATTGTTATATGCTCCATCTCTAATTGTATCTCTATATTTCGGTTTTGTAGGTACTTACCAATTGGAGTACTATCATTAAAGCTACATCAGAGTATCCAGCAGAGGAACTACATCAAGTGAATTTGTGCATCGACATAGCAATGCTTTGTGTGGATTCTGAAAGAGTCAATAGACCCACCATAGCTGGTATCCTAGATGCATTGAATAGGACAAAAACTCATATGCCCTCCTCTACGAAAAAAACTCATATTCCCTGGGGACAGGTATGATTTGCATACTTGCAAACAAAATGAAATCTCGAGTATATATTTGCAATCTGTAGAAGACAGTTGCTTGGATATATGGACCACTAAGTAGTTATAGAGTTTGCAGCTCCCCGTCTCCCACTCATTTTATTCTCAATCAAGTAGTTCTTTAATAGTCAGGAACTTGCTTACTGCATCCTTTTGACTCCCTGCTCTATAATCCATGTAGAAGAACCTTCATTTTAGTTCCGGCTAATTCCAGGAATAGAAAACTAGAGAGGGCCTATTCGTAATCGTGCCTTCCGGAGTGACAGGCTAAGTGAAGGGCAGGGGGATGCTGCCCTCGACAACCGTGGCTGTGATTGGCACTGTCGTGCTCATACGAGGTACCAGACGGTGTAGAAGTTAACCTAGTTGATTAATCTTAGGTGTGGTCATGCTAGATAGCTATATGAAAGAGCCATACATGTAGTTCAAGTAGTGCATGCAAGATTCCAACATTCAAAATCGTGCCTTGTACTATGGAAGGGGAAAGGGAGGGGTAACACGTAATGAGTGCCCTATAAGCCTTACACAATAGCTTTATCAGACCACTGTGGCGCCCTAACTGACGCCAACAGAGGTAGCTGCAATGGTTCGATGAGATAGCGGTGAGAGAGAAGGGGCAGGGGGACATTGGTGGCAGGTGTAAGGGAAAAAGGGAGAGGAGTGAAGCCGGCTGGGTACCTTGGTGGGGGAGAGGAAAGGGTGGAGGAAGAACAAAGAGGTGAGGCGCCTGCTAGTGATTGCACTGTAAGCCTACCGCGCGACATTGCTCCAAAGCTACGCTCTCCCAATAAAGGAGAACTTCTAGAGAGTTGATATGAATTAAAGAGATTACCACAGACTCACATAGTGCCTGAGGTATTAGCCACATTTCCTTTCATGCCCTTGCCGAGGGGCTTTCCTCGGCGCCTCTCACTTTGGGCTTTGCTTCTTCAAAGGTGGTGTTTAGGCCGCAAAGAGTACAACCAGTGTGTTATGTGTGTGCACTTTCGGTGTGTTACAATTTGCCATTATTGCTTGATGCTTTATTACTATTCAAAATAGTTTCTCTTTTTCCAAGTTGTCATTTTAACATAGCATTATAGATTTTGTCCTTCCGATTTGCATGTTTTGATCGTCTATAACTTAGTTTACATAATGGAAGCACATCCCAGAGAGTAAATTGATCATGAGATCTTGACCATGATGATTCTCCTGTTTTTTTCCTTGTACTTACACATAAAAGTTGTTTCAGTTGGAAGATGTGCCCCTGTGTTCGACAATTGGTCCCAAAAGTACGAGTAAAAGGTCGAACCCAGTTCCCACAAAGGAAAATAAAAGGTTGAAGATGATGACAACTGAAGTGGACAATATCGCGAACAAACACCAACAGTTTAATTGCATGCCAGGAGATAGCTCTAAAACTATTGTTCAGCAAGTTCCAGACAGGGAAACATCATCAGATGTGGAACCGACATTAATCATTGGAAGGGATGAAGAAAAACATAAAATATTGTCCATTTTATCTGAGAGCAACGCAGAAGAGATGACCATCCTTCCAATATATGGCATCGGAGGAATTGGCAAGACAACCTTGGCACAATTGGTGTTCAATGACATACAGTTCCGGGACTACTATCGGGTGTGGGTATATGTTTCTCAGAAGTTTGACTTAAAGAAAATTGGCAACTTTATAATATCACAGTTAACAAAAGAGACCAGCGATATAGATGACCAGCAGACACTTCATAATCGCCTTAGACAGCTATTTGCTGGTAAGAGTATCCTTATTGTTTTAGATGACCTGTGGGAGGAGAAACAACATGAGTTAGAGAAATTGAAGGCTATGCTAAGGCTTGGCATAGGAAACAAGGTTGTCATAGTAACTACACGTGATGAAGCCATTGCAAGGAAAATCAACAGGACTGTTATGCCATACAAGCTAGAGATTTTAACAGATGATATGTGCTGGTCTATAATAAAACAAAAAAGTTTCTTTGAAGATCGATGTGACAAAGAACAATTGGGGCAGATCGGAATGGACATTGCAATCAAGTGTGGAGGTGTGGCTTTGGCGGCTCAATCACTTGGGTACATGTTGAGGGAGATGGAGTCTGACCAATGGGAGTCAGTGAGGGACAGTTATATCTGGAATCTATCTACTATGGAAGATCCATCATTAAGAAATCATGAAGTGCTTCTGTCCTTGCTGTTAAGCTATTCCCATATGCATGAATTCTTGCAGTTATGCTTTTCCTATTGTGCATTCTTTCCAAAAGGTCAAAATATAGTGAAGTATGATCTAATTCACCAGTGGATAGCTCTTGGATTCACCGGTCCATCTGGAATATTTGATTCTATTCAGCTCTGTGAGAAATATATTACACGGCTTTTGGGGATGTCATTCCTTCAATATTCAAAGACACGTTCGGTGAGTTACTACATACTCTCGATGTCCCAAAAGATAGCTATGGGTAGTTTCTTCATGTCAAAGAGTCCCCTTCCAGTACTGCTAGGTGTCAGGTTTCTAGAAGGCCGCTAGGGCATCTTAGATAGGGTCATAGTTATACACTACTCATCCTCAAATGCATATGCCTGTGCAATTTTCTTTTCTAGATGACCTTCTCGACAAGCTCGTTGACATTTATCCTTTTTCTTTTTCTTTTCTTTCCCTTGTTTTCAACCTTACCTTTCAAATTTCCTTTTCCAAGAATGACATTCAAGTCCATAACCTGATCGTGGATATGGGTCCTACTAAAGCCCTTTGGAGCTCAATATTTTTCAACTATTTCATTAAAATGAATTCACATCTATAATCATCATTTCTTTTGTTATGTATGTATATAAAACAATACTAATTATTGTTGAACTAATAAACACATCGTTGATTACCTCTAAACAAATTTGAATGTCATTAAATTTGTCTTCATATTTTTTAGTGGGATAAGACCCCAATCCAACAGGCGCCCAAACAAATGGACCTATGTACTGAAACGTTGCTGTTGCTGGTGCATTTGTAGTGCTGGGTATTAATTTTAGCAGGTTTAAGATGAAAACCACTGCAGATATTTATCCCAGGCATTATTTCATTTGATATAAGCTTTGAAGTTTACAGATCCATAGTGTAATCTACTCTGGTGTAATTTAAATATACTGATCCGTTGCCCATTATCGAGAAAACATACAGCTACGGTTACACTCTTTTATAGTGATACAAAAGTATTTCTGTTGATAAAATATACTACTATAAAACAAAATAAATTCAATATTCTAACAACATTACGTGGTTTTGCTGCAGAGTGATGAACGGCAGGACAAAGATGTTAAAATGTTTGTAATGCATGACCTAGTGCACGATCTTGCAAGAGCAATATTGGCTGATAAAGTTAATAAAGAGGGTGATGCTGTGGGAAGCAGTTGTCACTATGCATTGCTCACAGATTGTAGCAAGCCATTGCAGTTGTCTGTTAGTTCAACTGAATATAGCCGGTTCAATTTTTTTCTTAGCCTGTTTAAAAAGAAGAGTTCACATGAAAATATAAAGGCGTTACGTTTTCTGAACTGTGGCAAAGTACTACTTCGCGGTGATGCATTTTCACCTGCCAAGTTCCTCCTTGTCTTAGATCTAAGTGAATGCTTTATTCAGAAGCTCTCACTTGATTCGATTGGACAACTGAGGCACTTGAGATATCTTTGTGCTCCACGGGTCAACGATTACACGATTCCCAACTGTATCACCAAGCTCTCAGAATTAACTTACCTCAACCTTAGAGGCTCTTGTCGTATCTCAGCATTGCCAGAGTCAATTGGCGATATGAAAAGTCTGATGCATCTTGATTTATCAGGCTGCTGTGACATAATTGAACTCCCAGTATCATTTGCGAAGCTGAAACAGTTGGTGCATCTAGATTTATCACACTGTCACGTGTCTGTATCAGAAGATTTTGGTGGCTTTACCAAACTTCAATATTTGAATTTATCAGTTTTGTTTAGTTCTTCCAAGGGGCATAGGAGAGGACTGCTAGAGGTCATTGGCAATTTAAAGAAACTCAGGTATCTAAATCTATCTCGGTGCATGGAGGACATAGCCACATCAGAAAACCAAATTGGCAGTTTGCTTGACTCTATCAGTACCCTTTCCAACCTTGAGCATCTGGACTTGTCTGAGAATAAACAGCTTTCCAGTATACCAGAAAGTATGGGCAACCTCAGGAAGCTTCATACATTGGACCTCTTAGGCTGCTATCAACTAGAGAAGCTTCCTGATAGTATGATTAATATGGTTAGCCTGAAGGTTCTAAATGTGGGTAATTTGGTTACACTGGATGAATCTGTGCTCTCTTTGTTAAATATTGCCTCCTTGCCACACTTTGTGGTGCATGCTTCAAGTGGTAAATGTAGCAGCAATATCACCCGTCTTCAGGCTACAAATCCTGATAGACTGATTATAGATAGACTTGAAAATGTCAAATCTGCAGAAGAGGCACATAACATAAAACTGATAGAGAAACAGAAAATTGAAACCCTACAATTTGAATGGACTGTGGCTGCTAGGAGGTTTGTGGATGACAAAGAGGTGTTGGAAAAACTAGTGCCGCCAAGCAGTGTCGACAGTTTGTGTATAATTGGTTATAGAAGTGTCAGCATTCCTGATTGGCTTCTGGGTATTAGTCAGTATCTCCCTAATCTTGCGATTATAAGTCTGGTTAATTTTTCTAAGTGCAAGAACCTACCACCACTCGGTCAACTACCAAACTTACAATGGCTGACTCTCAGCAGTATGGATGGTTTGGAGGAGTGGAACACGACATATACTACTGGAGAGCAAGGTAGAAACGAACTCTTGTTCCCTAAGCTTGAGAGATTAAACATACATGACTGTGCCAAGTTGAGGATAGAACCATGTCTGCCTAGAGCTTTGTATTTGCGCATACGAGATAGTAATAATGTGCTATCCTCACTCAATACAAGAGAGCAAGCTGAGAGCACGCTGCCCTCGGACATAGCACATTGTGATAATATGATATCAGCATGCGGAAAGAGTTCGTCATACAGCGGTGCTTCCTCTTCTTCTCCAATAACTGATCTGTTTGTAGAGGAAAGCAAACTACCCTTGCATCAGTGGAGGTTGCTTCACCAACTCCCCGCGCTCCGTGGTTTACGGATCAAACATTGCAGTGATCTGACCACCTCACTTGCTGTTATCCAAAAACTCTCCTCCCTCCAAAATTTGAGCCTGGAGCTCAACGACCATGAACTGCCGAGTTGGTTGATTCAGCTGACAGATCTACAGGAATTAAAGCTTATGCATTGCAATAGCATTACATCACTACCACAGTGGTTTGGAGAACTTGCATCTCTCAAGAGAATTGAGATCAAGTACTGCAAGGGGATCAGCTCTTTGCCGGAGAGCATACAACAACTGACTAAGCTTGAATTTCTAAGCATTCATGGCTGTCCTGTATTAGAGGAGTGGTGTGAATCAGAGGAGAACAAGATGAAGCTCACTCACATCAAAGTTGAGGTATGTGCGTGCAAGTTATCTGTTGTATTGCTTTTATTCTCGTGCTGGTAGTGACTTAATACTCTTTTCTTAAATGGCAAGTATACACATGCCATGAGTATCTTTACATAATCATGGTAAGTGTTGAATTAGGTGTATGTATTTTGTCTATTAGATGCTTCATGTGTCTAGATTACTTGACAAAAATATGTGACGACTGCATTAATAATTCGCCTAAGAAGAAAAGCATTCCAGTTGTGATTGTGCTATATCATGCACCTATACATGCATTGTTCTGATTATATATCCCGTTTGCATTGTTCAGATCGCTGGACGGGATTCGGTAGGCTTTGAGGATTCGAAGGTTCAGATTGTCAAACCAATGCCAGCACAAATGGTTCGCCAATCAGCATTTGCTACTACAGAACGAAGATAG

Example 4—HILAGE: Oat

Haploid inducer methods: Oat (Avena sativa) HILAGE-based methods areconducted with standard oat in vivo haploid induction using a cross witha maize pollen to pollinate an emasculated oat spike, embryo rescue intissue culture, and subsequent chromosome doubling techniques such asthose known in the art (see, e.g., Rines, “Oat haploids from widehybridization,” in Double Haploid Production in Crop Plants: A Manual,Maluszynski, Kasha, Forster and Szarejko (Eds.), pp. 155-159, KluwerAcademic Publishers, Dordrecht, Netherlands, 2003). Briefly, the in vivotechnique of oat haploid induction first requires that an emasculatedoat panicle be pollinated with maize pollen and treated with 2,4-D and50 mg/L gibberellic acid (GA3) two days after pollination. Fourteen daysafter pollination, the developing oat embryos are removed from the spikeand transferred to tissue culture. The developing embryo is grown intissue culture into a plantlet following methods described by Rines(supra). The plantlet is then chromosome doubled and transplanted to thesoil to produce doubled haploid seed.

Endonuclease transgene and transgenic construct: Oat HILAGE-basedmethods add the targeted mutagenesis component to the in vivo haploidinduction system and thus require an endonuclease. In oat HILAGE, one ormore of the maize chromosome(s) carry an endonuclease transgene capableof causing targeted double strand breaks in the wheat genome. Usefulendonucleases include, for example, meganucleases, ZFNs, TALE nucleases,and CRISPR/Cas-based endonuclease systems. The endonuclease is designedto target AsFAD2a and AsFAD2b, but an endonuclease could be designed totarget nearly any sequence. The endonuclease(s) are constructed usingmethods such as, without limitation, those described by Sander et al.(supra), Cermak et al. (supra), and Liang et al. (supra). The promoterused to drive expression of the endonuclease is endogenous or exogenous.High expression of the endonuclease is essential to increase the chancethat a targeted mutation is successful before the removal of the maizechromosomes carrying the endonuclease transgene. Suitable promoters areexpressed during early embryo development, and can be endogenous orexogenous. Examples are provided in TABLE 8.

The endonuclease construct also may include a selectable marker, such asa gene that confers herbicide resistance, to assist in recovery of thetransgene during whole plant transformation and subsequent backcrossing.When included, the herbicide resistance selectable marker is driven by apromoter with strong expression in maize and/or oat.

In another embodiment, the transgenic construct containing theendonuclease or a second construct combined into the same maize linecontains one or more copies of a sequence of DNA with homology to theDNA at and flanking the target site. This sequence of DNA may containnucleotide changes such as one or more base pair substitutions,deletions, and/or additions. Alternatively, this sequence may contain agene, a promoter, a regulatory sequence and or a transgene.

Testing the endonuclease in transgenic oat: While HILAGE-based methodsdo not use a transgenic oat line to generate the final product ofdoubled haploid oat with targeted mutations, it may be beneficial,though not necessary, to test the efficacy of the targeted endonucleaseconstruct in a transgenic oat line. Oat transformation could beconducted following techniques such as, but not limited to, thosedescribed by Zhang et al. (J. Plant Physiol. 148(6):667-671, 1996; andPlant Cell Reports, 18(12):959-966, 1999). Transgenic oat with putativemutations can be checked for targeted mutations using methods similar tothose described for Arabidopsis. Endonucleases demonstrating efficacyfor causing double stranded breaks are utilized for oat HILAGE-basedmethods.

Generating a maize line for oat HILAGE: A major difference between oatHILAGE-based methods and normal doubled haploid creation in oat is thata transgenic maize line is being used for haploid induction instead of aconventional maize line. As such, a maize line is being transformed withthe endonuclease construct. The endonuclease transgene could be added tothe haploid inducer using several methods such as, but not limited to:Agrobacteria methods (Gasparis et al., Plant Cell Reports27(11):1721-1729, 2008) or by particle bombardment (Somers et al.,Nature Biotechnol 10(12):1589-1594, 1992). Since the line used for maizetransformation likely is not a prolific haploid inducer, it may bebeneficial, though not necessary, to backcross the endonucleasetransgene(s) into a genetic background shown to be effective and highlyefficient at oat haploid induction. The backcross introgression of theendonuclease transgene into a more suitable maize line could beconducted with the assistance of molecular markers to select for thepresence of the transgene, as well as to select for the geneticbackground of the recurrent parent (the suitable maize line) and againstthe donor parent line (the originally transformed maize line).

Depending on the promoter chosen to drive the endonuclease, theendonuclease will likely show different expression in the maize linethan in the progeny of the maize-oat cross. If the gene is expected toexpress in maize, it may be beneficial to assess RNA and proteinexpression of the endonuclease to confirm that the endonuclease isfunctional.

Genotyping of mutated oat plants: The plantlets are genotyped before orafter transplanting to soil to identify (1) if the desired targetedmutation(s) occurred, (2) if the oat plant no longer contains maizechromosomes (a necessary test in oat), and (3) if the transgene(s) areno longer present. The third test is not necessary if the maizechromosomes are removed, but it is still probably a good standardoperating procedure to ensure removal of the transgene as there issignificant current market place resistance to transgenic oat).Additionally, potentially different tillers may need to be genotyped asthe plant could be chimeric for one or more targeted mutations and orfor the presence of maize chromosomes. Checking for mutation(s) at thetarget site(s) can be conducted as previously described for Arabidopsis.The presence of maize chromosomes, is assessed by one or more of severalmethods. For example, primers can be designed to amplify specificsequences on each of the 10 maize chromosomes in the maize line used forhaploid induction, and these primers can be used to determine if themaize chromosomes are still present. Alternatively, a custom SNP chipcan be designed that can be used to genotyped the oat line and alsomaize DNA. In oat plants that have lost the maize chromosomes, the oatSNPs are able to be genotyped, but the maize SNPs are not able to begenotyped. Alternatively or additionally, a low coverage whole genomesequencing method or RNA sequencing method could be utilized todetermine if the maize chromosomes are present and/or maize genes arebeing expressed. If the maize chromosomes have been removed from the oatplant, it is likely that the transgene had also been removed. However,to increase industry and consumer acceptance of HILAGE-based methods, itmay be beneficial to test for the absence of the transgene(s) in the oatline. In one method, primers that amplify portions or all of thetransgenic construct can be designed and used to test if any portion ofthe construct is in the produced oat line. Alternatively, the sequencesof the transgene can be search for in whole genome sequence or RNAsequence data, if said data are available.

Utilization of oat HILAGE: The maize line containing one or moreendonuclease and or CRISPR guide RNAs is being crossed (as the pollendonor) to an oat line to generate haploid progeny. Before the maizechromosomes are eliminated, the targeted endonuclease induces targetedDNA double strand breaks in the DNA from the oat line. Some of thesedouble stranded breaks will be incorrectly repaired and a mutation willresult. The haploid progeny genomes can be doubled before or after theprogeny are screened for the mutation(s). Once the genomes of thesehaploid individuals are doubled the individuals can be grown out andself-pollinated to produce doubled haploid seed. Different mutations maybe produced, and each mutation event is evaluated to determine if it hasthe desired result. Only lines with EM are advanced.

In some cases, HILAGE-based methods are conducted on all (or many) ofthe oat lines that may be used as parents for breeding. If populationsusing lines that have an EM at all targeted loci are developed, thepopulations will not segregate for the EM. Thus the breeding efforts aresimplified by not having to conduct selections for the presence of theEM.

Advantages of HILAGE-based methods in oat: The use of HILAGE could playa pivotal role in generating targeted mutations in oat. Globally, thereis still resistance to utilizing transgenes in oat. HILAGE-based methodsmay induce targeted mutations in oat without the released oat line evertechnically coming in contact with a transgene placed into an oatchromosome. In addition, HILAGE-based methods may be more effect in oatthan in maize since it is likely that the maize chromosomes persistlonger in the maize-oat embryo than the haploid inducer maizechromosomes persist in the maize haploid inducer-regular maize lineembryo. The additional time that the maize chromosomes are residing inthe oat embryo, the more opportunity for targeted mutations to occur.

Exemplary target sites and methods for genotypic screening in oat areprovided in TABLE 15A, while exemplary primers for amplifying the targetsite are provided in TABLE 15B. Expected genotypic classes from thecross are shown in TABLE 16.

Oat genes to target—AsFAD2: Sequences of the target oligonucleotideswere as follows (with f1/r1 for target 1 and f2/r2 for target 2).

AsFAD2 (SEQ ID NO: 36) f1 5′ GATTGGGTGCCGGTGGCAGGATGA 3′ (SEQ ID NO: 37) r1 5′ AAACTCATCCTGCCACCGGCACCC 3′ Underlining indicates the 20 bp target sequences.

TABLE 15A  Target Site and Methods for Genotypic Screening, Target 1Endonuclease CRISPR Gene Target AsFAD2 Gene Target AsFAD2Target sequence GGGTGCCGGTGGCAGGATGACGG  (SEQ ID NO: 38)Mutant Phenotype Increased oleic acid levels Genotypic ScreenCAPS assay; enzymes BtsCI,  BsrFI, BanI, and NlaIV

TABLE 15B  PCR primers for amplifying CRISPR target site,Target 1 & 2: AsFAD2a and AsFAD2b Gene Target AsFAD2 Forward Primer5′-TTCGTCCCGTCAACAAGAGG-3′  (SEQ ID NO: 39) Reverse Primer5′-GTCCGTCGGCGAGCGCTGG-3′  (SEQ ID NO: 40)

TABLE 16 Expected genotypic classes from cross Due to the method of oathaploidization formed from crossing emasculated oat panicles with maizepollen, the self-pollination outcome class is not very likely to occur.If the oat is emasculated correctly, no self-pollination should occur.If a mistake is made in the emasculation process and an oat seed isallow to self-pollinate, the seed will grow more vigorously than an oatx maize cross, and the seed can be easily screened out. Due to theability of oat and maize chromosomes to pair and the inability of maizechromosomes to be inherited, classes B and C are possible, andphenotyping and genotyping need to be done to remove these classes.Haploid Identification Successful Phenotype Trait: Mutation indicatesClass (mixed options) Trait: plant is: A Screened out visual NaSelf-pollination as a healthy seed B Visual phenotypic NA Hybrid, notassessment and homozygous genotypic mutated evaluation with molecularmarkers C Visual phenotypic NA Hybrid, is mutated assessment andgenotypic evaluation with molecular markers D Slow growing Wild-typeHaploid, not embryo, tests levels of seed homozygous negative for maizeoleic acid mutated DNA E Slow growing Higher Haploid, mutated embryo,tests levels of negative for maize seed DNA oleic acidPutative Consensus Sequence for AsFAD2 (and Surrounding Sequence)(This sequence was identified by aligning oat contigs that putativelycode for FAD2. Oat contigs found by searching for alignments in Oat COREdatabase that have high sequence similarity to barley, rice, and maizeFAD2's (5′UTR+coding). ATG start is bolded and underlined. Putative FAD2gene is underlined.

(SEQ ID NO: 41) CATAAACCACTCGTTCGTCCCGTCAACAAGAGGAGCAGAGGCGAGGGACTCGCGCTCGCGTGTGTGGTGTCCTTCCCTCGATCTGCCCCTCTCCGGCCAGTTCTAT CACCTCCTATCAGCAACATGGGTGCCGGTGGCAGGATGACGGAGAAGGAGAGGGAGAAGCAGGAGCAGCTCGGCCGCGCCGACGTCGGTGCGACCCTCCAGCGCTCGCCGACGGACAAGCCGCCGTTCACACTGGGGCAGATCAAGAAGGCGATCCCACCCCACTGCTTCCAGCGCTCGGTGATCAAGTCATTCTCCTACGTGGTCCATGACCTCGTCATCGTGGCTGCTCTCCTGTACGCCGCGCTGGTCTGGATCCCCACCCTCCCGAGCGTGCTGCAGCTGGGCGCCTGGCCGCTCTACTGGATCGTGCAGGGCTGCGTCATGACCGGCGTCTGGGTCATCGCGCACGAGTGCGGCCACCACGCCTTCTCCGACTACTCGCTCCTCGACGACATCGTCGGCCTGGTGCTCCACTCGTGGCTGCTGGTCCCGTACTTCTCGTGGAAGTACAGCCACCGTCGCCACCACTCCAACACCGGCTCCATGGAGCGTGACGAGGTGTTCGTCCCCAAGCAGAAGGACGCGCTGGCCTGGTACACCCCATACATCTACAACAACCCCATCGGCCGTCTGGTGCACATCGTGGTGCAGCTCACCCTCGGGTGGCCGCTGTACCTGTCGATGAACGCCTCGGGCCGCCCGTACGCGCGCTTCGCCTGCCACTTCGACCCCTACGGCCCCATCTACAACGACCGGGAGCGCGTCCAGATCTTCATTTCGGACGTCGGTGTGGTGGCCACGGCGTTCACCCTCTTCAAGCTTGCTTCGGCGTTCGGGTTCTGGTGGGTGGTGCGCATCTACGGTGTGCCGCTGCTGATCGTGAACGCGTGGCTGGTCCTGATCACCTACCTGCAGCACACCCACCCGGCGCTGCCGCACTACGACTCCACCGAGTGGGACTGGCTGCGGGGGGCGCTGGCCACCATGGACCGGGACTACGGCATCCTCAACCGCGTGTTCCACAACATCACGGACACGCACGTGGCGCACCACCTCTTCTCCACCATGCCGCACTACCATGCCATGGAGGCCACCAAGGCGATCAAGCCAATCCTGGGCGAGTACTACCAGTTCGACCCCACCCCCGTGGCCAAGGCAACATGGCGCGAGGCCAAGGAGTGCATCTACGTCGCGCCCACCGAGGACCGCAAGGGCGTCTTCTGGTACAGCAACAAGTTCTAGATTCGTCATGGGGACCTGCTGTGCTGCTGGAATGTGAGGAGGAAGAAGTCAGTAATACACCAAGTATCCATCCATCTACCTACATATGGTTGGGGGTTAGTAGTCTTTAGATAGAAGAGAGCGTTGTTTGGGCACAAGGAAAAGACTATGACCACCGTGCCAATGCTAGAAGAGTCGAAGCAGGTGCAACGAGGAGTAGCGTGTCGGGTGTCCGTGGCTTTGGTCAGTTCCGTCCTGTGTCTTTACTTCCTAGTCGCCGGTTT 

Example 5—HILAGE: Barley, Using Crosses to Hordeum bulbosum or Maize(Zea maize)

Haploid inducer methods: Barley (Hordeum vulgare) HILAGE-based methodsare conducted using the standard barley in vivo haploid induction usinga cross with a Hordeum bulbosum or maize line, embryo rescue techniques,and subsequent chromosome doubling techniques such as, withoutlimitation, those described by Kasha and Kao (Nature 225:874-876, 1970),Chen and Hayes (Theor. Appl. Genet. 77:701-704, 1989), Chen et al.(Genome 34:603-605, 1991), Laurie and Bennett (“Chromosome behavior inwheat x maize, wheat x sorghum and barley x maize crosses,” In KewChromosome Conference Proceedings III, Brandham (Ed.), Norwich, UK: TheStationery Office Books, pp. 167-177, 1988), and others. Briefly, the invivo technique of barley haploid induction first requires that a crossbe made between Hordeum vulgare and Hordeum bulbosum or maize, thehaploid inducer line. In HILAGE-based methods, the haploid inducer stockline is likely used as the male, since for the barley haploid inductionmethod, the Hordeum bulbosum or maize is used as the pollen donor andthe female is the Hordeum vulgare. The in vivo technique for barleyhaploid induction first requires that a Hordeum vulgare plant beemasculated and then pollinated by Hordeum bulbosum or maize, thehaploid inducer line. The developing barley embryos are removed from thespike and transferred to tissue culture. The developing embryo is grownin tissue culture into a plantlet, chromosome doubled, and grown tomaturity to produce doubled haploid seed following methods described by,for example, Kasha and Kao (supra).

Endonuclease transgene and transgenic construct: Barley HILAGE-basedmethods add the targeted mutagenesis component to the in vivo haploidinduction system, and thus require an endonuclease. Endonuclease areconstructed using methods such as those described by Sander et al.(supra), Cermak et al. (supra), and Liang et al. (supra). Examples ofsuitable endonucleases include, without limitation, meganucleases, ZFNs,TALE nucleases, and CRISPR/Cas-based nucleases. The endonuclease isdesigned to target Vrs1, but an endonuclease can be designed to targetnearly any sequence. The promoter used to drive expression of theendonuclease is endogenous or exogenous. High expression of theendonuclease is essential during the first couple stages of mitosis inthe developing embryo. Suitable promoters are expressed during earlyembryo development, and can be endogenous or exogenous. Examples areprovided in TABLE 8.

The endonuclease construct may also include a selectable marker, such asherbicide resistance to assist in recovery of the transgene during wholeplant transformation and subsequent backcrossing. The selectable markeris not required for HILAGE-based methods and thus, in anotherembodiment, the endonuclease construct does not have a selectable markerfor recovery during transformation.

In another embodiment the transgenic construct containing theendonuclease or a second construct combined into the same barley linecontains one or more copies of a sequence of DNA with homology to theDNA at and flanking the target site. This sequence of DNA may containnucleotide changes such as one or more base pair substitutions,deletions, and/or additions. Alternatively, this sequence may contain agene, a promoter, a regulatory sequence and or a transgene.

Testing the endonuclease in transgenic barley: It may be beneficial,though not necessary, to test the efficacy of the targeted endonucleaseconstruct in a transgenic barley line. Barley transformation isconducted according to techniques such as those described by, withoutlimitation, Tingay et al. (Plant J., 11(6):1369-1376, 1997) and Travellaet al. (Plant Cell Reports, 23(12):780-789, 2005). Transgenic barleywith putative mutations also may be checked for targeted mutations usingmethods similar to those described for Arabidopsis herein.Endonuclease(s) showing efficacy at causing double stranded breaks areutilized for barley HILAGE-based methods.

Introgression of the endonuclease transgene into the haploid inducer:The next step in barley HILAGE-based methods is the addition of atransgenic endonuclease gene to the Hordeum bulbosum or maize haploidinducer line. The endonuclease transgene could be added to the haploidinducer using several methods. One method involves the directtransformation of the haploid inducer to add the transgene, using, forexample, Agrobacteria methods as described by Tingay et al. (supra),Travella et al. (supra), and Ishida et al. (supra), or particlebombardment as described by Travella et al. (supra) and Gordon-Kamm etal. (supra). Alternatively, a line amenable to transformation can firstbe transformed with the endonuclease transgene, and then this line withthe endonuclease transgene can be crossed to a haploid inducer line. F₁diploid progeny from the cross can be screened, and can be backcrossedto the haploid inducer line. This backcrossing process can be repeatedseveral times to recover the majority of the haploid inducer's geneticswith the addition of the endonuclease transgene. After a sufficientnumber of backcrosses (e.g., two, three, or four backcrosses), theresulting backcross plant (BC₃F₁ following three backcrosses) can beself-pollinated to produce BC₃F₂ individuals. The BC₃F₂ individuals canbe screened to find individuals that are genetically very similar to thehaploid inducer line and are homozygous for the endonuclease transgene.In the second method, molecular markers could be used to selectbackcross individuals that contain the transgene and contain highpercentages of the haploid inducer genome. These selected individualscan be used for the next round of backcrossing to more quickly recoverthe genome of the haploid inducer with the addition of the endonucleasetransgene. The resulting line that functions as a haploid inducer lineand contains the endonuclease transgene is designated as the haploidinducer stock line.

Testing expression of the endonuclease transgene: Following eitherdirect transformation or transformation of another line followed bybackcrossing, several tests are run to evaluate expression of theendonuclease in the haploid inducer stock line. Alternatively,expression tests are conducted before or concurrently with thebackcrossing to select transgenic events with high expression.Specifically, expression assays for RNA and for protein of theendonuclease transgene can be conducted to insure that the transgene iscorrectly being expressed. Transformation events with higher expressionare desired for HILAGE-based methods. Efficacy of the transgene andtransformation event can additionally be assessed by determining ifmutations are detected in the target site(s) of the line. The presenceof mutations can be evaluated as described herein for Arabidopsis.Events with high gene expression and the presence of mutations in thetarget site(s) can be outcrossed to targeted lines to identify ifhaploid progeny with mutations are generated. Desirable haploidinducer-transgenic event combinations produce a high frequency andnumber of haploid progeny with targeted mutations.

Utilization of Barley HILAGE: The Hordeum bulbosum or maize haploidinducer is crossed as the male to the Hordeum vulgare to generatehaploid progeny. The haploid progeny genomes can be doubled before orafter the progeny are screened for the mutation(s). Once the genomes ofthese haploid individuals are doubled the individuals can be grown outand self-pollinated to produce doubled haploid seed. It may be necessaryto genotype multiple tillers per plant as the plant could be chimericfor one or more targeted mutations. Different mutations may be produced,and evaluation of each mutation event is necessary to determine if themutation(s) obtained have the desired result. EM that produce thedesired phenotype (e.g., a mutation that causes a frame shift andeliminates proper gene function) are advanced.

In some embodiments, HILAGE-based methods are conducted on all (or many)of the barley lines intended for use as parents for breeding. Ifpopulations are developed using lines that have an EM at all targetedloci, the populations will not segregate for the EM. Thus the breedingefforts are simplified by not having to conduct selections for thepresence of the EM.

Exemplary target sites and methods for genotypic screening in oat areprovided in TABLE 17A, while exemplary primers for amplifying the targetsite are provided in TABLE 17B. Expected genotypic classes from a crosswith Hordeum bulbosum are shown in TABLE 18A, and expected genotypicclasses from a cross with maize are shown in TABLE 18B.

Barley gene to target—Vrs1 (BAF43315.1): Sequences of the targetoligonucleotides were as follows (with f1/r1 for target 1 and f2/r2 fortarget 2).

Barley Vsr1 gene (SEQ ID NO: 42) f1 5′ GATTGGCGGAGGGGATGGTGACGG 3′ (SEQ ID NO: 43) r1 5′ AAACCCGTCACCATCCCCTCCGCC 3′ 

Underlining indicates the 20 bp target sequences.

TABLE 17A  Target Site and Methods for Genotypic Screening, Target 1Endonuclease CRISPR Gene Target Vrs1 Gene Target Protein ID BAF43315.1Target sequence GGCGGAGGGGATGGTGACGGTGG  (SEQ ID NO: 44)Mutant Phenotype Change from 2 row to 6 row spikes Genotypic ScreenCAPS assay; enzyme HpyCH4III  and Tsp45I

TABLE 17B  PCR primers for amplifying CRISPR target site, Target 1Gene Target: Barley Vrs1 Forward Primer 5′-TCCAACGTGGACACGACTTT-3′ (SEQ ID NO: 45) Reverse Primer 5′-GAGGTGGCATTTGTGGAGGA-3′ (SEQ ID NO: 46)

TABLE 18A Expected genotypic classes from cross: Vrs1 endonucleasetarget (Hordeum bulbosum haploid inducer) Haploid inducer stock line isthe male. Most regenerated plants from tissue culture are haploid (>95%)Interspecific hybrids (diploid plants from the cross between Hordeumvulgare and Hordeum bulbosum) can be recognized by their abnormal growthhabit and presence of pubescence on leaf sheaths (trait from Hordeumbulbosum parent) (Devaux, “The Hordeum bulbosum (L.) method,” in DoubleHaploid Production in Crop Plants: A Manual, Maluszynski, Kasha,Forster, and Szarejko (Eds.), pp. 15-19, Dordrecht, Netherlands: KluwerAcademic Publishers.). Haploid Identification Successful PhenotypeTrait: Mutation indicates Class various Trait: plant is: A Developingseed will be na Self-pollination larger for self-pollinations BInterspecific hybrids na Hybrid, not recognized by abnormal homozygousgrowth habit and mutated presence of pubescence on leaf sheaths (traitfrom Hordeum bulbosum parent) C Interspecific hybrids na Hybrid, isrecognized by abnormal mutated growth habit and presence of pubescenceon leaf sheaths (trait from Hordeum bulbosum parent) D Weak plant Spikeis Haploid, not still 2 row homozygous mutated E Weak plant Spike changeHaploid, to 6 row mutated

TABLE 18B Expected genotypic classes from cross: Vrs1 endonucleasetarget (maize haploid inducer) Haploid inducer stock line is the male.Most regenerated plants from tissue culture are haploid (>90%) (Chen etal., supra) Haploid Identification Successful Phenotype Trait: Mutationindicates Class various Trait: plant is: A Developing seed naSelf-pollination will be larger for self-pollinations B na na Hybrid,not homozygous mutated C na na Hybrid, is mutated D Weak plant Spike isHaploid, not still 2 row homozygous mutated E Weak plant Spike changeHaploid, to 6 row mutated

Hordeum vulgare subsp. vulgare Vrs1 gene for homeodomain leucine zipper protein Vrs1, complete cds, allele: Vrs1.b. GenBank: AB259782.1. The translation start site is underlined, and exon sequences are bolded. (SEQ ID NO: 47)GTCATAACTCGGCAAACATAGATTAGACAGAATTTTCTGAGTTCTTATCTAGAGGAACTCGATGAACTTGAGGCATTGTCGAGGTTCTTCCTTTCACCGAGTACTTTTTTGCGTGTACTAGGCAAATATATGAAGTTTGTGAGTTTCGGATCACCACCGAGTGCAAGTTTGGACCAAACTTGACAAATACATAAGTTTGGCGAGCTCCGAATGAAATGAACTCTGCAAAAGAATAGAACTCGGCGCAAAACCAGATTCTAATAGTGTGTGAATTTTTGGGCTGTTTTGTATAAATATGATGAAACTTAGTAAAATTTCACTCAGGTCAATGCTAATGTGGAGAGTAAATAAAAAATGAAGGGAGTACTTGGCTGCATCATATGTTTGCCCCCGATCACCTTCACATCTCCCCGTCCGGACGGCCTGGATCGGAAAGCACTCAGCCGGAGCCCCGCCGGCGCTTGCCGTTGGGTACCTCTGCCACCTATTTATATTACCCCTAGGTCTCTCCCTGGAGACACGCACTCCCCTCCTTCAACTAGTGCTTTGCGGCCCGTGGTCCTCCTCTCGATCCAGTTCCTGAGCACACCAACAGGCAACAGAACAACCTACCGTGTCTCCCCTCCAATCTCCTCACGATCCCTTCTTTCCCTCAGATCCGAACCGAAAGC ATG GACAAGCATCAGCTCTTTGATTCATCCAACGTGGACACGACTTTCTTCGCGGCCAATGGTACACACGACGCCGCGCGCGCCCGGTCTTTGCGCATGCGATGATGCAGCTGCAGTAGCTTCAGTTTCACCGGCCAGGACACGCATGTGATGACGTTTTTTCCATTCTGTGTTTGTATGTGCAGGCACGGCGCAGGGGGATACCAGCAAGCAGAGGGCGCGGCGCAGGCGGCGGAGGTCGGCGAGGTGCGGCGGAGGGGATGGTGACGGTGGGGAGATGGACGGAGGAGGGGACCCCAAGAAGCGGCGGCTCACCGACGAGCAGGCCGAGATTCTGGAGCTGAGCTTCCGGGAGGACCGCAAGCTGGAGACAGCCCGCAAGGTGTATCTGGCCGCCGAGCTCGGGCTGGACCCCAAGCAGGTCGCCGTGTGGTTCCAGAACCGCCGCGCGCGCCACAAGAACAAGACGCTCGAGGAGGAGTTCGCGAGGCTCAAGCACGCCCACGACGCCGCCATCCTCCACAAATGCCACCTCGAGAACGAGGTATGCTTGCTCGCATACACTCACACTGGCTTACATATGGCGCTGCACATCTGCAGTTCCTCTCCGTTCTTGAACATGCTTACTGACAAACATATGGCCAGCTGCTGAGGCTGAAGGAGAGACTGGGAGCGACTGAGCAGGAGGTGCGGCGCCTCAGGTCGGCAGCTGGGAGCCACGGGGCATCTGTGGATGGCGGACACGCCGCTGGCGCCGTTGGCGTGTGCGGCGGGAGCCCGAGCTCGTCCTTCTCGACGGGAACCTGCCAGCAGCAGCCGGGTTTCAGCGGGGCAGACGTGCTGGGGCGGGACGATGACCTGATGATGTGCGTCCCCGAGTGGTTTTTAGCATGAATTAGAGTTTATGCTGGCTAAGCCGATAGCAGCGTGGTCGAGTGTTTTTTAGCATGAAATCAGATCTCCATCTCCCATAAAATAGCCGAGATAGCTGCTGCCGCCGCCAAATCCTCTATAGGGCTTCAAGATCGGCAGAAACCTCTAGAAATCATCTCCCCCCTCCGGAAAAGTCGCCTCTATTTGTCTCCATTGCCCGCGATGCAGCATCCGGTATAGCTGCTAAGACAGGCCGCCCCTAAATCGTTTCTCCAGCGATTTTAATCTTTGGTTTTTAGCCTGTATATATGGGCTGTGATTTGAAGTTGAGACGAGCTGGACATCAACTGCACGCTGATCGATTACTATTCTAGTTTGGCATAGTGTTAATTAAGTTTGGATGATCTCTAGGCGTGCGTTAAGTATGTAGATAGTGTTGATTAATGGCAAAAGCTTGCAAGTTAAGTGTAGTATTGGCAGCTCTCTTGAAGATCAAATATGATGTGTGTTATCATTTGATGATATATATTTTACTTCAGCCGTAAATAGTCTTCTTAGGGAAGCACTGTCCATGTATGTGCTGGTAGTTGGCATTCATCTTTC 

Example 6—HILAGE: Triticale

Haploid inducer methods: Hexaploid triticale (×Triticosecale Wittm.)HILAGE-based methods are conducted with standard triticale in vivohaploid induction using a cross with a maize pollen to pollinate anemasculated triticale spike, embryo rescue in tissue culture, andsubsequent chromosome doubling techniques such as, but not limited to,those described by Wedzony et al. (“Factors influencing triticaledoubled haploid production by means of crosses with maize,” In:Proceedings of the 4^(th) International Triticale Symposium, Red Deer,Canada. Vol 1. Juskiw (Ed.) International Triticale Association,Alberta, Canada, pp. 45-52, 1998; and Plant Breed. 117:211-215, 1998),Wedzony (“Protocol for doubled haploid production in hexaploid triticale(×Triticosecale Wittm.) by crosses with maize,” In Double HaploidProduction in Crop Plants: A Manual, Maluszynski, Kasha, Forster, andSzarejko (Eds.), pp. 135-140, Dordrecht, Netherlands: Kluwer AcademicPublishers, 2003), and others. Briefly, the in vivo technique oftriticale haploid induction first requires that an emasculated triticalespike be pollinated with maize pollen and treated with Dicamba(3,6-dichloro-2-methoxybenzoic acid) 1-2 days after pollination.Subsequently (18-21 days after pollination), the developing triticaleembryos are removed from the spike and transferred to tissue culture.The plant is eventually transplanted to the greenhouse and treated withcolchicine to double the chromosome number, and doubled haploid seed isharvested.

Endonuclease transgene and transgenic construct: Triticale HILAGE-basedmethods add the targeted mutagenesis component to the in vivo haploidinduction system, and thus require an endonuclease. Examples of suitableendonucleases include, but are not limited to, meganucleases, ZNFs, TALEnucleases, and CRISPR/Cas-based nucleases. The endonuclease is designedto target Tsn1, but an endonuclease can be designed to target nearly anysequence. The Tsn1 gene was brought into triticale on the wheat 5BLchromosome. Thus, the description of the Tsn1 CRISPR/Cas target sites,primers, etc., described below are identical to those used for targetingTsn1 in wheat as described herein. The endonuclease(s) are constructedusing methods such as, without limitation, those described by Sander etal. (supra), Cermak et al. (supra), and Liang et al. (supra). Thepromoter used to drive expression of the endonuclease is endogenous orexogenous. High expression of the endonuclease is essential to increasethe chance that a targeted mutation is successful before the removal ofthe maize chromosomes carrying the endonuclease transgene. Suitablepromoters are expressed during early embryo development, and can beendogenous or exogenous. Examples are provided in TABLE 8.

The endonuclease construct also may include a selectable marker, such asherbicide resistance, to assist in recovery of the transgene duringwhole plant transformation and subsequent backcrossing. When present,the herbicide resistance selectable marker is driven by a promoter withstrong expression in maize and or triticale. The selectable marker isnot required for HILAGE-based methods, however.

In some embodiments, the transgenic construct containing theendonuclease or a second construct combined into the same maize linecontains one or more copies of a sequence of DNA with homology to theDNA at and flanking the target site. This sequence of DNA may containnucleotide changes such as one or more base pair substitutions,deletions, and/or additions. Alternatively, this sequence may contain agene, a promoter, a regulatory sequence and or a transgene.

Testing the Endonuclease in transgenic triticale: While HILAGE-basedmethods do not use a transgenic triticale line to generate the finalproduct of doubled haploid triticale with targeted mutations, it may bebeneficial, though not necessary, to test the efficacy of the targetedendonuclease construct in a transgenic triticale or transgenic wheatline. Triticale transformation is conducted following techniques suchas, without limitation, those described by Zimny et al. (MolecularBreeding, 1(2):155-164, 1995). Wheat transformation is conductedaccording to techniques such as those described by Weeks et al. (PlantPhysiol. 102(4):1077-1084, 1993). Transgenic triticale with putativemutations is assessed for targeted mutations using methods similar tothose described in for the Arabidopsis herein. Endonucleases showingefficacy for causing double stranded breaks are utilized for triticaleHILAGE-based methods.

Generating a maize line to use for triticale HILAGE: One majordifference between triticale HILAGE-based methods and normal doubledhaploid creation in triticale is that a transgenic maize line is beingused for haploid induction instead of a conventional maize line. Assuch, a maize line is being transformed with the endonuclease construct.The endonuclease transgene is added to the haploid inducer using any ofseveral methods, including Agrobacterium-based methods (e.g., thosedescribed by Ishida et al., supra) or by particle bombardment (such asthe method described by Gordon-Kamm et al., supra). Since the line usedfor maize transformation likely is not a prolific haploid inducer, itmay be beneficial, though not necessary, to backcross the endonucleasetransgene(s) into a genetic background that has previously shown highefficacy in triticale haploid induction. The backcross introgression ofthe transgene into a more suitable maize line may be conducted with theassistance of molecular markers to select for the presence of theendonuclease transgene, as well as to select for the genetic backgroundof the recurrent parent (the suitable maize line) and against the donorparent line (the originally transformed maize line).

Depending on the promoter chosen to drive the endonuclease, theendonuclease will likely show different expression in the maize linethan in the progeny of the maize-triticale cross. If the gene isexpected to express in maize, it may be beneficial to assess the RNA andprotein expression of the endonuclease to confirm that the endonucleaseis functional.

Genotyping of putative mutated triticale plants: The plantlets are beinggenotyped before or after transplanting to soil to identify (1) if thedesired targeted mutation(s) occurred (2) if the triticale plant nolonger contains maize chromosomes and (3) if the transgene(s) are nolonger present. Additionally, potentially different tillers may need tobe genotyped as the plant could be chimeric for one or more targetedmutations. Assays to evaluate the presences of mutation(s) at the targetsite(s) can be conducted as described in the Arabidopsis section herein.The presence of maize chromosomes, could be assessed by one or more ofseveral methods. Primers can be designed to amplify specific sequenceson each of the 10 maize chromosomes in the maize line used for haploidinduction, and these primers can be used to determine if the maizechromosomes are still present. Alternatively, a custom SNP chip can bedesigned that can be used to genotyped the triticale line and also maizeDNA. In triticale plants that have lost the maize chromosomes, thetriticale SNPs are able to be genotyped, but the maize SNPs are not ableto be genotyped. Alternatively or additionally, a low coverage wholegenome sequencing method or RNA sequencing method could be utilized todetermine if the maize chromosomes are present and/or maize genes arebeing expressed. If the maize chromosomes have been removed from thetriticale plant, it is likely that the transgene had also been removed.However, to increase industry and consumer acceptance of HILAGE-basedmethods, it may be beneficial to test for the absence of thetransgene(s) in the triticale line. In one method, primers that amplifyportions or all of the transgenic construct can be designed and used totest if any portion of the construct is in the produced triticale line.Alternatively, the sequences of the transgene can be search for in wholegenome sequence or RNA sequence data, if said data are available.

Utilization of Triticale HILAGE: The maize line containing one or moreendonuclease and or CRISPR guide RNAs is being crossed (as the pollendonor) to a triticale line to generate haploid progeny. Before the maizechromosomes are eliminated, the targeted endonuclease induces targetedDNA double strand breaks in the DNA from the triticale line. Some ofthese double stranded breaks will be incorrectly repaired and a mutationwill result. The haploid progeny genomes can be doubled before or afterthe progeny are screened for the mutation(s). Once the genomes of thesehaploid individuals are doubled, the individuals can be grown out andself-pollinated to produce doubled haploid seed. Different mutations maybe produced, and evaluation of each mutation event is necessary todetermine if the mutation(s) obtained will have the desired result. OnlyEM that produce a desired phenotype are advanced.

In some embodiments, HILAGE-based methods are conducted on all (or many)of the triticale lines that may be used as parents for breeding. Ifpopulations are developed using lines that have an EM at all targetedloci, the populations will not segregate for the EM. Thus, breedingefforts are simplified by not having to conduct selections for thepresence of the EM.

Advantages of HILAGE in triticale: HILAGE may play a pivotal role ingenerating targeted mutations in triticale. Globally, there is stillresistance to utilizing transgenes in triticale. HILAGE-based methodsmay induce targeted mutations in triticale without the releasedtriticale line ever technically coming in contact with a transgeneplaced into a triticale chromosome. In addition, it is possible thatHILAGE-based methods may be more effective in triticale than in maize,since it is likely that the maize chromosomes persist longer in themaize-triticale embryo than the haploid inducer maize chromosomespersist in the maize haploid inducer-regular maize line embryo. Thelonger the maize chromosomes are present in the triticale embryo, themore opportunity for targeted mutations to occur.

Exemplary target sites and methods for genotypic screening in oat areprovided in TABLE 19A, while exemplary primers for amplifying the targetsite are provided in TABLE 19B. Expected genotypic classes from thecross are shown in TABLE 20. Triticale (wheat) gene to target—Tsn1:Sequences of the target oligonucleotides were as follows (with f1/r1being for target 1 and f2/r2 being for target 2):

Tsn1 gene (SEQ ID NO: 30) f1 5′ GATTGCCGCTAGGGCATCTTAGAT 3′ (SEQ ID NO: 31) r1 5′ AAACATCTAAGATGCCCTAGCGGC 3′ 

Underlining indicates the 20 bp target sequences.

TABLE 19A  Target Site and Methods for Genotypic Screening, Target 1Endonuclease CRISPR Gene Target Triticale (Wheat) Tsn1 Gene TargetADH59425 Target sequence GCCGCTAGGGCATCTTAGATAGG  (SEQ ID NO: 32)Mutant Phenotype Resistance to Stagonospora nodorum,which causes Stagonospora nodorum blotch (SNB); and resistance toPyrenophora tritici-repentis,  which causes tan spot.* Genotypic CAPS assay; enzymes SfaNI, DdeI,  Screen CAPS BglI, TauI, and AciI*Faris et al., Proc. Natl. Acad. Sci. U.S.A. 107(30):13544-13549, 2010.

TABLE 19B  PCR primers for amplifying CRISPR target site, Target 1Gene Target Triticale (Wheat) Tsn1 Forward Primer5′-TGTGCATTCTTTCCAAAAGGTCA-3′  (SEQ ID NO: 33) Reverse Primer5′-GCTCCAAAGGGCTTTAGTAGGA-3′ (SEQ ID NO: 34)

TABLE 20 Expected genotypic classes from cross: Endonucleases targetedto Tsn1 Due to the method of triticale haploidization formed from thecrossing of emasculated triticale spikes with maize pollen, severalclasses of plant outcomes are not possible. If the triticale isemasculated correctly, no self- pollinations should occur. If a mistakeis made in the emasculation process and a triticale seed is allow toself-pollinate, the seed will grow more vigorously than a triticale xmaize cross, and the seed can be easily screened out. Due to theinability of triticale and maize chromosomes to pair and the inabilityof maize chromosomes to be inherited, classes B and C are not possible.Thus, only classes D and E are expected to be produced. HaploidSuccessful Identification Mutation Phenotype Trait: embryo Trait:disease indicates Class growth resistance plant is: A Screened out naSelf-pollination visual as a healthy seed B NA na Hybrid, not homozygousmutated C NA na Hybrid, is mutated D Slow growing Susceptible Haploid,not embryo to SNB homozygous mutated E Slow growing Resistant Haploid,mutated embryo to SNB

Triticale (wheat) Tsn1 and surrounding sequence (GENBANK ® accession number GU259618) (SEQ ID NO: 35)ATGACTACACCAATGAGTATACCGTTCGCAACTTTGGAAAAGATTACAAATGGGTTCTCAAACGATTTAATAATTGGAAGGGGTGGGTATGGAAACGTTTACAAGGTATGGCTTAATACTTGATATTTCCTTTTTTCAGCAAATGTTCAGGCTATAAACAAATAATTTAAGTGCAATAATTATGTCAAGCAGGCAGTTTACAAAGGGGAAGTGATTGCTGTGAAGTTGCTTCATGATGATCTGGTGCAATTACTTGATGACAGACAATTTAAAAATGAACTTTTTAACCTTTTGAGGGTTGAGCATCCGAATATTGTTTGCTTACGTGGTTATTGTTATGAAACACGGTATAAAATTGTTAAGCACAATGGTGAGACAGTCTTTGGTAAACATATACACAGAGTTCTCTGCTTTGAATACTTGGAGGGTGGAAGCCTAGACAATCATCTTCATGGTACGATGGAACTTCAAAATACAGTTATTTTGTTTTACGTTTAAAGGAAACTGATTTCTCATTTACATACATACTCTTTGTTAACTTGCGTAGCACCATCTTTGCCACCTAACTGGACCACACGTTACAATACCATAAAGGGGATTTGTGAAGGCTTAAATTTCCTTCACGGATGTCAACCACCAATTTTGCATCTTGATCTGAAGCCTGCCAATATATTAGTAGACAGTTCCATGGTGCCTAAACTGGCGGATTTTGGATTGTCAAAGCTCTTCCATGGATCACATACTCATGTGACAAAACAAATCATAGGAACCCAGTAAGCGGAAGCGACCCGTGGATTGTCTCGTTCTGAATTTTCTTTCTTTTGTGATCAAATAAATAGTATGTACAGTTCTGTACTAACTGTGTCTTTGTATCACGCAGGAAGTACATGCCACCGGAATTCATCAAAGATGGCAAGATCTCGGTTAAAAATGATGTCTTTAGTTTGGGTATTGTGATCATAGAAATAATGGCAGGACCTATGGGTTATTCAGAATTTTCAGAAATGGGCAGCGGTGCACAATTTGTGAAGGAGGTAATAAAAAAAACTCAAGTTTGACACCCGAGTTCGTATAAATAACAAACTACCACACCAAGAATTTGATGTCTAATGTGTGAGCCATTATAATCGTTGAACTGAGTTTATGACAGGACCGGCAGTAATAAAAAATATAGCAACACTCCCCCACACAATATATTGAGCATAGAAGATACAACTTATCTAGCTATAACAAAATAATAATCCAGAAAAGTAGCCATTTTTTTTTCCGGACAGGATTGAGGTCCACCAGTCCAATAACTATGAAGCAGCTCGCTGATAGAAAATTCCAAGGTACAATTATTTTTGTAAGTTTCTCCTTATCACGTGTGAAACACCAATGTAATAAAGCTGATAAACCAAACGTACCCACTATGAGAACTGCATACACTGAGACTCGAAGAAAAGAACAAATGCATATCTAGAACCTTGCTCCATGGGATATCTAGAACCTTGCTCCATGGGATCTAGCACCATCTCCATTTTGGAGCAAGCACGAGGTGCGTATCGTAATCTTTTTCTGCTAGATGCAGACTTAGACACCCAGTATTCTCTAGGTAAATTATTTATCTGGAAAGTCGTAGGTAACACTTGTGAACAAGGATATAGCGTACATATATATGGGAGCATTTGTGTTATGTGACACTTTTGACTTAATTGCAAATATTATGTTATGTGAAGACTCAAGAGTGTTTTTGAACAAGTATCGTACATATTGTACCGAAAAAGGCTTTCGCCCCGCTTTATATTATAAAGCACATGCCCAAGCCAACAAACCACACAGGTTCACAAACACACGCAGACCCACACACACCAAGTTCACACACAGACAAGATCCACAAGGGTTAATGCTGAGGGCACAGCTTAACAAGCCCTAGAACAAAAAGGAAAGACACCATCTAGTCGGGCTCCGGGGGGGGGGGGGGGGGGGGCGGCGGAAGTGGAGGCGCCAGGCGGAAGGCGAGCGATCGAAGGTCGGCGAGGAGGGTGTTGATGATGTCCCGATCCTGAGGGCGGCTAAGCGGCCGCCAAAGCTGCAAGTACCCACACATTTTAAAAATGGCGTCAGTAGCGCGTCGTAGAGGGACTTTTTGGATGACAAGCTTATTGCGGACGGTCCACAGCGTCCAGCCGAGAACCCCAACGCATAACCAACGGATATGTCGGTGGCGTGGGGGGGGAGGCGTGGATTTCCGCGAGGAGGTCGGGGAAGTTGGAGTTGCACCACTATCCGCCAACCGTCTCACGGAAACTGGACCAAAGAAACTGGCCGCAGGGCACGTGAAGAAGATGTGGTTAGCATCCTCCGCAGTGCCGCACAAGGGGCAAAGCCCATCCCCGGGTCCGTTGCGCTTGAGGACTTCGACACCGGAGGGGAGGCGGCCACGAATCCACTGCCAAAGGAAGATCCTAATCTTCAGAGGTAAGCGAATGTCCCAGATCAGAGCAAAGGGCTCGGGCGCGGGCGAAGGCGCAATAGCCGCGTACATGACCTAGTAGAGAAACGACCGGAGGACTCTAGGCGCCACGAGATGGCGTCCGGGGCGTCGGTGACGCTCATCGGAAGAAGGGCGATGTCCTGGAGGAGGGAATCCCAGGCGGCCACTTCGGGGGGACCGAAAGGACGACGAAACGCGAGGCGCCCTAAGTCAATAAGGGCCGTCTCGACAGAGACCCGAGGGTCAACCGCAATGGTGAAGAGATCGGGAAAGCGGGCGGCCAGAGGGGTGTCACCGAGCCACCGATCAAACCAGAACAGGGTCGCGGACCCAGTACCAATCGAAATGGACGTGCCGATACGAAGCACAGGAAGCAGCCGCACGACGGCCTGCCAAAACTGTGATCCGCCCGAACGCTGACAGAAAGCCAGAGGCTGGCCACGGAGGTATTTGTTGCGGATAATGGTGAGCCACAACCCTCCGTCACCATTGGCAATACGCCACAACCACCGGGTCAGGAGGGCGATGTTCATTCGGCGGGAGGACAGAATCTCAAGACCCCCCTGGTCTTTAGGTTTACAAATGTCCGGCCAAGTCACCATGTGGTACTTCTGTTTGTCATCGTCGCCAGCCCAATAGAACCTGGATTGGTACTTGGCAATTTCCGTGTGCAGCGTTTCATGGAGGCTATAAAAGCTCATGAGGAACCAAAGGAGACTGGCGAGTGAGGAGTTGATGAGGATCACCCGCGCCGCCTTTGATAGCCAACGCCCTTTCCAAGGTTCGACGCGGTGTTGCATACGGGTCACCGTAGGGTGGAGGTCCGCCACGGTGAGGCGCGAGTCACTAACGGGGATCCCCAGGTAGGTCGTGGGGAAGGACCCTAGCCGACAGTTCAGGCGATCAGCAATATCCTGAGCCTCCTCCGGAGGGTATCCAAGGACCATCACCTCGCTCTTATCAAAGTTAATCGTAAGGCCCGACATCTGCTGGAAGCACAGGAGGAGGAACTTCAGGTTAGCAACATCCTGATTTGAACCTTCCACCATTATTATGGTGTCGTTCGCGTATTGCAGGAGGGAGACCCCTCCCCCTCCAACTAGGTGAGGGACAATGCCGTGGATATGGCCAGCACCCTTAGCCTTATCCAGGATGGCGGCCAGAGCATCGACCACCATGTTGAACAGGAACGGCGAGAATGGGTCTCCTGACAGACCCCACAGAGGGTGGGGAAGTATGGCCCAATCTCGCCGTTAATGTTCACCGCCGTCTTTCCACATGAAACTGATTGCATCACGCGGGTCACCCAGCGGTCATCAAAGCCCTTACGCAGCAGTACTTCCCGAAGGAAGGGCCAGTGAACAGTATCATAGGCTTTATGGAAGTCAAGCTTCAGGAACACAGCACGAAGATGCTTCACCCGGACCTCGTGAAGGACTTCATGGAATACCAACACGCCATCAAGAATAAACCGGCCTTGGATGAAGGCCGATTGGTTCGGGTGAGTGATCGAATCAGCCAGCAGGGTCACCCTATTGGCGTACCCTTTGGCCAGGATCCGAAAAATCACGTTAATCACCGTGATGGGGCGGAACTGGCGAATATCAGAGGCACCCGGAACCTTTGGGATGAGGGTAATGATCCCATAGTTGAGGCGTCCCAGGTCCATCGAACCCGAATAGAACTCCTCGAACAAAGCCATGACCTCCGGTTTGACCGCCTGCCAGAATGTTTTAAAGAAAGCAACAGGCAGGCCATCCGGGCCTGGGGCCGAGGCGGGGTTCATGCCTTTAATGGCCGCGAGCACCTCGTCCTCGGCGAAGGGAGCAACCAGGGCCGCATTGGCCTCGCCGGGAACCAACTGCGCCCCCGTCCAAGTATCGGGGGCATCGTACATATTGTTATATGCTCCATCTCTAATTGTATCTCTATATTTCGGTTTTGTAGGTACTTACCAATTGGAGTACTATCATTAAAGCTACATCAGAGTATCCAGCAGAGGAACTACATCAAGTGAATTTGTGCATCGACATAGCAATGCTTTGTGTGGATTCTGAAAGAGTCAATAGACCCACCATAGCTGGTATCCTAGATGCATTGAATAGGACAAAAACTCATATGCCCTCCTCTACGAAAAAAACTCATATTCCCTGGGGACAGGTATGATTTGCATACTTGCAAACAAAATGAAATCTCGAGTATATATTTGCAATCTGTAGAAGACAGTTGCTTGGATATATGGACCACTAAGTAGTTATAGAGTTTGCAGCTCCCCGTCTCCCACTCATTTTATTCTCAATCAAGTAGTTCTTTAATAGTCAGGAACTTGCTTACTGCATCCTTTTGACTCCCTGCTCTATAATCCATGTAGAAGAACCTTCATTTTAGTTCCGGCTAATTCCAGGAATAGAAAACTAGAGAGGGCCTATTCGTAATCGTGCCTTCCGGAGTGACAGGCTAAGTGAAGGGCAGGGGGATGCTGCCCTCGACAACCGTGGCTGTGATTGGCACTGTCGTGCTCATACGAGGTACCAGACGGTGTAGAAGTTAACCTAGTTGATTAATCTTAGGTGTGGTCATGCTAGATAGCTATATGAAAGAGCCATACATGTAGTTCAAGTAGTGCATGCAAGATTCCAACATTCAAAATCGTGCCTTGTACTATGGAAGGGGAAAGGGAGGGGTAACACGTAATGAGTGCCCTATAAGCCTTACACAATAGCTTTATCAGACCACTGTGGCGCCCTAACTGACGCCAACAGAGGTAGCTGCAATGGTTCGATGAGATAGCGGTGAGAGAGAAGGGGCAGGGGGACATTGGTGGCAGGTGTAAGGGAAAAAGGGAGAGGAGTGAAGCCGGCTGGGTACCTTGGTGGGGGAGAGGAAAGGGTGGAGGAAGAACAAAGAGGTGAGGCGCCTGCTAGTGATTGCACTGTAAGCCTACCGCGCGACATTGCTCCAAAGCTACGCTCTCCCAATAAAGGAGAACTTCTAGAGAGTTGATATGAATTAAAGAGATTACCACAGACTCACATAGTGCCTGAGGTATTAGCCACATTTCCTTTCATGCCCTTGCCGAGGGGCTTTCCTCGGCGCCTCTCACTTTGGGCTTTGCTTCTTCAAAGGTGGTGTTTAGGCCGCAAAGAGTACAACCAGTGTGTTATGTGTGTGCACTTTCGGTGTGTTACAATTTGCCATTATTGCTTGATGCTTTATTACTATTCAAAATAGTTTCTCTTTTTCCAAGTTGTCATTTTAACATAGCATTATAGATTTTGTCCTTCCGATTTGCATGTTTTGATCGTCTATAACTTAGTTTACATAATGGAAGCACATCCCAGAGAGTAAATTGATCATGAGATCTTGACCATGATGATTCTCCTGTTTTTTTCCTTGTACTTACACATAAAAGTTGTTTCAGTTGGAAGATGTGCCCCTGTGTTCGACAATTGGTCCCAAAAGTACGAGTAAAAGGTCGAACCCAGTTCCCACAAAGGAAAATAAAAGGTTGAAGATGATGACAACTGAAGTGGACAATATCGCGAACAAACACCAACAGTTTAATTGCATGCCAGGAGATAGCTCTAAAACTATTGTTCAGCAAGTTCCAGACAGGGAAACATCATCAGATGTGGAACCGACATTAATCATTGGAAGGGATGAAGAAAAACATAAAATATTGTCCATTTTATCTGAGAGCAACGCAGAAGAGATGACCATCCTTCCAATATATGGCATCGGAGGAATTGGCAAGACAACCTTGGCACAATTGGTGTTCAATGACATACAGTTCCGGGACTACTATCGGGTGTGGGTATATGTTTCTCAGAAGTTTGACTTAAAGAAAATTGGCAACTTTATAATATCACAGTTAACAAAAGAGACCAGCGATATAGATGACCAGCAGACACTTCATAATCGCCTTAGACAGCTATTTGCTGGTAAGAGTATCCTTATTGTTTTAGATGACCTGTGGGAGGAGAAACAACATGAGTTAGAGAAATTGAAGGCTATGCTAAGGCTTGGCATAGGAAACAAGGTTGTCATAGTAACTACACGTGATGAAGCCATTGCAAGGAAAATCAACAGGACTGTTATGCCATACAAGCTAGAGATTTTAACAGATGATATGTGCTGGTCTATAATAAAACAAAAAAGTTTCTTTGAAGATCGATGTGACAAAGAACAATTGGGGCAGATCGGAATGGACATTGCAATCAAGTGTGGAGGTGTGGCTTTGGCGGCTCAATCACTTGGGTACATGTTGAGGGAGATGGAGTCTGACCAATGGGAGTCAGTGAGGGACAGTTATATCTGGAATCTATCTACTATGGAAGATCCATCATTAAGAAATCATGAAGTGCTTCTGTCCTTGCTGTTAAGCTATTCCCATATGCATGAATTCTTGCAGTTATGCTTTTCCTATTGTGCATTCTTTCCAAAAGGTCAAAATATAGTGAAGTATGATCTAATTCACCAGTGGATAGCTCTTGGATTCACCGGTCCATCTGGAATATTTGATTCTATTCAGCTCTGTGAGAAATATATTACACGGCTTTTGGGGATGTCATTCCTTCAATATTCAAAGACACGTTCGGTGAGTTACTACATACTCTCGATGTCCCAAAAGATAGCTATGGGTAGTTTCTTCATGTCAAAGAGTCCCCTTCCAGTACTGCTAGGTGTCAGGTTTCTAGAAGGCCGCTAGGGCATCTTAGATAGGGTCATAGTTATACACTACTCATCCTCAAATGCATATGCCTGTGCAATTTTCTTTTCTAGATGACCTTCTCGACAAGCTCGTTGACATTTATCCTTTTTCTTTTTCTTTTCTTTCCCTTGTTTTCAACCTTACCTTTCAAATTTCCTTTTCCAAGAATGACATTCAAGTCCATAACCTGATCGTGGATATGGGTCCTACTAAAGCCCTTTGGAGCTCAATATTTTTCAACTATTTCATTAAAATGAATTCACATCTATAATCATCATTTCTTTTGTTATGTATGTATATAAAACAATACTAATTATTGTTGAACTAATAAACACATCGTTGATTACCTCTAAACAAATTTGAATGTCATTAAATTTGTCTTCATATTTTTTAGTGGGATAAGACCCCAATCCAACAGGCGCCCAAACAAATGGACCTATGTACTGAAACGTTGCTGTTGCTGGTGCATTTGTAGTGCTGGGTATTAATTTTAGCAGGTTTAAGATGAAAACCACTGCAGATATTTATCCCAGGCATTATTTCATTTGATATAAGCTTTGAAGTTTACAGATCCATAGTGTAATCTACTCTGGTGTAATTTAAATATACTGATCCGTTGCCCATTATCGAGAAAACATACAGCTACGGTTACACTCTTTTATAGTGATACAAAAGTATTTCTGTTGATAAAATATACTACTATAAAACAAAATAAATTCAATATTCTAACAACATTACGTGGTTTTGCTGCAGAGTGATGAACGGCAGGACAAAGATGTTAAAATGTTTGTAATGCATGACCTAGTGCACGATCTTGCAAGAGCAATATTGGCTGATAAAGTTAATAAAGAGGGTGATGCTGTGGGAAGCAGTTGTCACTATGCATTGCTCACAGATTGTAGCAAGCCATTGCAGTTGTCTGTTAGTTCAACTGAATATAGCCGGTTCAATTTTTTTCTTAGCCTGTTTAAAAAGAAGAGTTCACATGAAAATATAAAGGCGTTACGTTTTCTGAACTGTGGCAAAGTACTACTTCGCGGTGATGCATTTTCACCTGCCAAGTTCCTCCTTGTCTTAGATCTAAGTGAATGCTTTATTCAGAAGCTCTCACTTGATTCGATTGGACAACTGAGGCACTTGAGATATCTTTGTGCTCCACGGGTCAACGATTACACGATTCCCAACTGTATCACCAAGCTCTCAGAATTAACTTACCTCAACCTTAGAGGCTCTTGTCGTATCTCAGCATTGCCAGAGTCAATTGGCGATATGAAAAGTCTGATGCATCTTGATTTATCAGGCTGCTGTGACATAATTGAACTCCCAGTATCATTTGCGAAGCTGAAACAGTTGGTGCATCTAGATTTATCACACTGTCACGTGTCTGTATCAGAAGATTTTGGTGGCTTTACCAAACTTCAATATTTGAATTTATCAGTTTTGTTTAGTTCTTCCAAGGGGCATAGGAGAGGACTGCTAGAGGTCATTGGCAATTTAAAGAAACTCAGGTATCTAAATCTATCTCGGTGCATGGAGGACATAGCCACATCAGAAAACCAAATTGGCAGTTTGCTTGACTCTATCAGTACCCTTTCCAACCTTGAGCATCTGGACTTGTCTGAGAATAAACAGCTTTCCAGTATACCAGAAAGTATGGGCAACCTCAGGAAGCTTCATACATTGGACCTCTTAGGCTGCTATCAACTAGAGAAGCTTCCTGATAGTATGATTAATATGGTTAGCCTGAAGGTTCTAAATGTGGGTAATTTGGTTACACTGGATGAATCTGTGCTCTCTTTGTTAAATATTGCCTCCTTGCCACACTTTGTGGTGCATGCTTCAAGTGGTAAATGTAGCAGCAATATCACCCGTCTTCAGGCTACAAATCCTGATAGACTGATTATAGATAGACTTGAAAATGTCAAATCTGCAGAAGAGGCACATAACATAAAACTGATAGAGAAACAGAAAATTGAAACCCTACAATTTGAATGGACTGTGGCTGCTAGGAGGTTTGTGGATGACAAAGAGGTGTTGGAAAAACTAGTGCCGCCAAGCAGTGTCGACAGTTTGTGTATAATTGGTTATAGAAGTGTCAGCATTCCTGATTGGCTTCTGGGTATTAGTCAGTATCTCCCTAATCTTGCGATTATAAGTCTGGTTAATTTTTCTAAGTGCAAGAACCTACCACCACTCGGTCAACTACCAAACTTACAATGGCTGACTCTCAGCAGTATGGATGGTTTGGAGGAGTGGAACACGACATATACTACTGGAGAGCAAGGTAGAAACGAACTCTTGTTCCCTAAGCTTGAGAGATTAAACATACATGACTGTGCCAAGTTGAGGATAGAACCATGTCTGCCTAGAGCTTTGTATTTGCGCATACGAGATAGTAATAATGTGCTATCCTCACTCAATACAAGAGAGCAAGCTGAGAGCACGCTGCCCTCGGACATAGCACATTGTGATAATATGATATCAGCATGCGGAAAGAGTTCGTCATACAGCGGTGCTTCCTCTTCTTCTCCAATAACTGATCTGTTTGTAGAGGAAAGCAAACTACCCTTGCATCAGTGGAGGTTGCTTCACCAACTCCCCGCGCTCCGTGGTTTACGGATCAAACATTGCAGTGATCTGACCACCTCACTTGCTGTTATCCAAAAACTCTCCTCCCTCCAAAATTTGAGCCTGGAGCTCAACGACCATGAACTGCCGAGTTGGTTGATTCAGCTGACAGATCTACAGGAATTAAAGCTTATGCATTGCAATAGCATTACATCACTACCACAGTGGTTTGGAGAACTTGCATCTCTCAAGAGAATTGAGATCAAGTACTGCAAGGGGATCAGCTCTTTGCCGGAGAGCATACAACAACTGACTAAGCTTGAATTTCTAAGCATTCATGGCTGTCCTGTATTAGAGGAGTGGTGTGAATCAGAGGAGAACAAGATGAAGCTCACTCACATCAAAGTTGAGGTATGTGCGTGCAAGTTATCTGTTGTATTGCTTTTATTCTCGTGCTGGTAGTGACTTAATACTCTTTTCTTAAATGGCAAGTATACACATGCCATGAGTATCTTTACATAATCATGGTAAGTGTTGAATTAGGTGTATGTATTTTGTCTATTAGATGCTTCATGTGTCTAGATTACTTGACAAAAATATGTGACGACTGCATTAATAATTCGCCTAAGAAGAAAAGCATTCCAGTTGTGATTGTGCTATATCATGCACCTATACATGCATTGTTCTGATTATATATCCCGTTTGCATTGTTCAGATCGCTGGACGGGATTCGGTAGGCTTTGAGGATTCGAAGGTTCAGATTGTCAAACCAATGCCAGCACAAATGGTTCGCCAATCAGCATTTGCTACTACAGAACGAAGATAG

Example 7—HILAGE-HR

To conduct HILAGE-HR, an endonuclease is generated to cause a doublestrand break in a specific sequence. Initial studies utilize anendonuclease targeting maize Bm3 ZEAMMB73_595664, using the same CRISPRtarget sequences described above:

(SEQ ID NO: 19) f1 5′ GATTGGGCTCCACCGCCGGCGACG 3′ (SEQ ID NO: 20)r1 5′ AAACCGTCGCCGGCGGTGGAGCCC 3′

Underlining indicates the 20 bp target sequences.

The donor template consists of three fragments of DNA that aresynthesized into a single fragment in order. The first fragment is a DNAsequence homologous to the sequence upstream of the target site, and canrange in size from 10 nucleotides to 1,000 or more nucleotides. Thesecond fragment is the sequence GGGCCCGGCGACG (SEQ ID NO:49), whichcontains a 7 bp deletion relative to the wild-type and causes a frameshift mutation. The third fragment is DNA sequence that is homologous tothe DNA sequence downstream of the target site, and can range in sizefrom 10 nucleotides to 1,000 or more nucleotides.

One or more copies of the endonuclease and one or more copies of thedonor template are placed into the genome of a maize haploid inducerline, either by directly transforming the inducer line with thesequences or by first transforming the sequences into a different maizeline and backcrossing the sequences into the inducer line. The haploidinducer line with the endonuclease(s) and the donor template(s) line iscalled the HILAGE-HR line.

The HILAGE-HR line is crossed to an elite line having a sequence thatmatches or closely matches the endonuclease target sequence. TheHILAGE-based process is conducted as described herein, with themodification that in some individuals, the DSB is repaired using thedonor template sequence. The specific DNA modifications produced by theDSB being repaired using the donor template are detected using methodssuch as PCR and sequencing. The chromosome-doubled plants are screenedto identify plants that contain the desired insertion and do not containchromosomes from the haploid inducer line.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for generating a doubled haploid plantcell comprising a transgenic DNA sequence inserted at or near a selectedDNA sequence, said method comprising: (a) transforming a haploid inducerline with (i) a first transgenic DNA sequence flanked on both sides byDNA sequences homologous to sequences upstream and downstream of theselected DNA sequence, and (ii) a second transgenic sequence thatencodes a rare-cutting endonuclease, to generate a Haploid Inducer Linefor Accelerated Genome Editing (HILAGE) stock line having the first andsecond transgenic DNA sequences stably integrated therein, wherein thetransgenic DNA sequence encoding the rare-cutting endonuclease isoperably linked to a promoter that is expressed in plant embryos duringat least the first and second cell divisions after fertilization, andwherein the rare-cutting endonuclease is targeted to the selected DNAsequence; (b) crossing the HILAGE stock line with a targeted line togenerate an F₁ zygote comprising the stably integrated first and secondtransgenic DNA sequences; (c) culturing the F₁ zygote such that (i) therare-cutting endonuclease is expressed and cleaves chromosomal DNA ofthe targeted line at or near the selected DNA sequence, and the firsttransgenic DNA sequence is inserted at the site of cleavage, and (ii)genome elimination takes place such that chromosomes from the HILAGEstock line are eliminated, resulting in a haploid cell; and (d) inducingchromosome doubling in the haploid cell to generate a doubled haploidplant cell comprising the first transgenic DNA sequence but not thesecond transgenic DNA sequence.
 2. The method of claim 1, wherein theplant cell is from maize, wheat, barley, triticale, Arabidopsis, oat,pennycress, tomato, potato, soybean, or camelina.
 3. The method of claim1, wherein the rare-cutting endonuclease is a TALE endonuclease, aCRISPR/Cas-based nuclease, a ZFN, or a meganuclease.
 4. The method ofclaim 1, wherein the promoter is a cauliflower mosaic virus doubledenhanced 35S promoter, a maize ZmUb1 promoter, or a rice APX, OsCc1,EIF5, R1G1B, PGD1, Act1, or SCP1 promoter.
 5. A method for generating adoubled haploid plant cell comprising a first transgenic DNA sequenceinserted at or near a selected DNA sequence, said method comprising: (a)transforming a plant cell line with (i) a first transgenic DNA sequencethat is flanked on both sides by DNA sequences homologous to sequencesupstream and downstream of the selected DNA sequence, and (ii) a secondtransgenic DNA sequence that encodes a rare-cutting endonuclease, togenerate a transgenic plant cell line having the first and secondtransgenic DNA sequences stably integrated therein, wherein thetransgenic DNA sequence encoding the rare-cutting endonuclease isoperably linked to a promoter that is expressed in plant embryos duringat least the first and second cell divisions after fertilization, andwherein the rare-cutting endonuclease is targeted to the selected DNAsequence; (b) crossing the transgenic plant cell line to a haploidinducer line to generate a HILAGE stock line that is homozygous for thefirst and second transgenic DNA sequences, has the first and secondtransgenic DNA sequences stably integrated therein, and has the majorityof its DNA from the haploid inducer line, where the HILAGE stock linecan induce haploids upon crossing; (c) crossing the HILAGE stock linewith a targeted line to generate an F₁ zygote comprising the stablyintegrated first and second transgenic DNA sequences; (d) culturing theF₁ zygote such that (i) the rare-cutting endonuclease is expressed andcleaves chromosomal DNA of the targeted line at or near the selected DNAsequence, and the first transgenic DNA sequence is inserted at the siteof cleavage, and (ii) genome elimination takes place such thatchromosomes from the HILAGE stock line are eliminated; and (e) inducingchromosome doubling in the haploid cell to generate a doubled haploidplant cell comprising the first transgenic DNA sequence but not thesecond transgenic DNA sequence.
 6. The method of claim 5, wherein theplant cell is from maize, wheat, barley, triticale, Arabidopsis, oat,pennycress, tomato, potato, soybean, or camelina.
 7. The method of claim5, wherein the rare-cutting endonuclease is a TALE endonuclease, aCRISPR/Cas-based nuclease, a ZFN, or a meganuclease.
 8. The method ofclaim 5, wherein the promoter is a cauliflower mosaic virus doubledenhanced 35S promoter, a maize ZmUb1 promoter, or a rice APX, OsCc1,EIF5, R1G1B, PGD1, Act1, or SCP1 promoter.
 9. A method for generating adoubled haploid plant cell comprising a transgenic DNA sequence insertedat or near a selected DNA sequence, said method comprising: (a) crossinga HILAGE stock line with a targeted line to generate an F₁ zygotecomprising a stably integrated transgenic DNA sequence, wherein theHILAGE stock line comprises (i) a stably integrated first transgenic DNAsequence flanked on both sides by DNA sequences homologous to sequencesupstream and downstream of the selected DNA sequence, and (ii) a stablyintegrated second transgenic DNA sequence that encodes a rare-cuttingendonuclease, wherein the transgenic DNA sequence encoding therare-cutting endonuclease is operably linked to a promoter that isexpressed in plant embryos during at least the first and second celldivisions after fertilization, and wherein the rare-cutting endonucleaseis targeted to the selected DNA sequence; (b) culturing the F₁ zygotesuch that (i) the rare-cutting endonuclease is expressed and cleaveschromosomal DNA of the targeted line at or near the selected DNAsequence, and the first transgenic DNA sequence is inserted at the siteof cleavage, and (ii) genome elimination takes place such thatchromosomes from the HILAGE stock line are eliminated; and (c) inducingchromosome doubling in the haploid cell to generate a doubled haploidplant cell comprising the first transgenic DNA sequence but not thesecond transgenic DNA sequence.
 10. The method of claim 9, wherein theplant cell is from maize, wheat, barley, triticale, Arabidopsis, oat,pennycress, tomato, potato, soybean, or camelina.
 11. The method ofclaim 9, wherein the rare-cutting endonuclease is a TALE endonuclease, aCRISPR/Cas-based nuclease, a ZFN, or a meganuclease.
 12. The method ofclaim 9, wherein the promoter is a cauliflower mosaic virus doubledenhanced 35S promoter, a maize ZmUb1 promoter, or a rice APX, OsCc1,EIF5, R1G1B, PGD1, Act1, or SCP1 promoter.